A non-essential amino acid occurring in natural form as the L-isomer. It is synthesized from GLYCINE or THREONINE. It is involved in the biosynthesis of PURINES; PYRIMIDINES; and other amino acids.
The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.
A non-essential amino acid. In animals it is synthesized from PHENYLALANINE. It is also the precursor of EPINEPHRINE; THYROID HORMONES; and melanin.
A group of enzymes that catalyzes the phosphorylation of serine or threonine residues in proteins, with ATP or other nucleotides as phosphate donors.
A family of enzymes that catalyze the conversion of ATP and a protein to ADP and a phosphoprotein.
Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.
Electron transfer through the cytochrome system liberating free energy which is transformed into high-energy phosphate bonds.
The phosphoric acid ester of serine.
The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.
Established cell cultures that have the potential to propagate indefinitely.
An essential amino acid occurring naturally in the L-form, which is the active form. It is found in eggs, milk, gelatin, and other proteins.
Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.
Protein kinases that catalyze the PHOSPHORYLATION of TYROSINE residues in proteins with ATP or other nucleotides as phosphate donors.
An serine-threonine protein kinase that requires the presence of physiological concentrations of CALCIUM and membrane PHOSPHOLIPIDS. The additional presence of DIACYLGLYCEROLS markedly increases its sensitivity to both calcium and phospholipids. The sensitivity of the enzyme can also be increased by PHORBOL ESTERS and it is believed that protein kinase C is the receptor protein of tumor-promoting phorbol esters.
Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.
A group of enzymes that are dependent on CYCLIC AMP and catalyze the phosphorylation of SERINE or THREONINE residues on proteins. Included under this category are two cyclic-AMP-dependent protein kinase subtypes, each of which is defined by its subunit composition.
An amino acid that occurs in endogenous proteins. Tyrosine phosphorylation and dephosphorylation plays a role in cellular signal transduction and possibly in cell growth control and carcinogenesis.
The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.
Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.
A protein-serine-threonine kinase that is activated by PHOSPHORYLATION in response to GROWTH FACTORS or INSULIN. It plays a major role in cell metabolism, growth, and survival as a core component of SIGNAL TRANSDUCTION. Three isoforms have been described in mammalian cells.
A superfamily of PROTEIN-SERINE-THREONINE KINASES that are activated by diverse stimuli via protein kinase cascades. They are the final components of the cascades, activated by phosphorylation by MITOGEN-ACTIVATED PROTEIN KINASE KINASES, which in turn are activated by mitogen-activated protein kinase kinase kinases (MAP KINASE KINASE KINASES).
The phosphoric acid ester of threonine. Used as an identifier in the analysis of peptides, proteins, and enzymes.
The rate dynamics in chemical or physical systems.
Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.
Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.
The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES.
Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases.
A ubiquitous casein kinase that is comprised of two distinct catalytic subunits and dimeric regulatory subunit. Casein kinase II has been shown to phosphorylate a large number of substrates, many of which are proteins involved in the regulation of gene expression.
Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility.
A PROTEIN-TYROSINE KINASE family that was originally identified by homology to the Rous sarcoma virus ONCOGENE PROTEIN PP60(V-SRC). They interact with a variety of cell-surface receptors and participate in intracellular signal transduction pathways. Oncogenic forms of src-family kinases can occur through altered regulation or expression of the endogenous protein and by virally encoded src (v-src) genes.
The intracellular transfer of information (biological activation/inhibition) through a signal pathway. In each signal transduction system, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) is mediated via the coupling of a receptor/enzyme to a second messenger system or to an ion channel. Signal transduction plays an important role in activating cellular functions, cell differentiation, and cell proliferation. Examples of signal transduction systems are the GAMMA-AMINOBUTYRIC ACID-postsynaptic receptor-calcium ion channel system, the receptor-mediated T-cell activation pathway, and the receptor-mediated activation of phospholipases. Those coupled to membrane depolarization or intracellular release of calcium include the receptor-mediated activation of cytotoxic functions in granulocytes and the synaptic potentiation of protein kinase activation. Some signal transduction pathways may be part of larger signal transduction pathways; for example, protein kinase activation is part of the platelet activation signal pathway.
A group of enzymes removing the SERINE- or THREONINE-bound phosphate groups from a wide range of phosphoproteins, including a number of enzymes which have been phosphorylated under the action of a kinase. (Enzyme Nomenclature, 1992)
Products of proto-oncogenes. Normally they do not have oncogenic or transforming properties, but are involved in the regulation or differentiation of cell growth. They often have protein kinase activity.
The parts of a macromolecule that directly participate in its specific combination with another molecule.
A proline-directed serine/threonine protein kinase which mediates signal transduction from the cell surface to the nucleus. Activation of the enzyme by phosphorylation leads to its translocation into the nucleus where it acts upon specific transcription factors. p40 MAPK and p41 MAPK are isoforms.
Proteins and peptides that are involved in SIGNAL TRANSDUCTION within the cell. Included here are peptides and proteins that regulate the activity of TRANSCRIPTION FACTORS and cellular processes in response to signals from CELL SURFACE RECEPTORS. Intracellular signaling peptide and proteins may be part of an enzymatic signaling cascade or act through binding to and modifying the action of other signaling factors.
A CALMODULIN-dependent enzyme that catalyzes the phosphorylation of proteins. This enzyme is also sometimes dependent on CALCIUM. A wide range of proteins can act as acceptor, including VIMENTIN; SYNAPSINS; GLYCOGEN SYNTHASE; MYOSIN LIGHT CHAINS; and the MICROTUBULE-ASSOCIATED PROTEINS. (From Enzyme Nomenclature, 1992, p277)
Phosphotransferases that catalyzes the conversion of 1-phosphatidylinositol to 1-phosphatidylinositol 3-phosphate. Many members of this enzyme class are involved in RECEPTOR MEDIATED SIGNAL TRANSDUCTION and regulation of vesicular transport with the cell. Phosphatidylinositol 3-Kinases have been classified both according to their substrate specificity and their mode of action within the cell.
A glycogen synthase kinase that was originally described as a key enzyme involved in glycogen metabolism. It regulates a diverse array of functions such as CELL DIVISION, microtubule function and APOPTOSIS.
The level of protein structure in which combinations of secondary protein structures (alpha helices, beta sheets, loop regions, and motifs) pack together to form folded shapes called domains. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure. Small proteins usually consist of only one domain but larger proteins may contain a number of domains connected by segments of polypeptide chain which lack regular secondary structure.
Proteins prepared by recombinant DNA technology.
A 44-kDa extracellular signal-regulated MAP kinase that may play a role the initiation and regulation of MEIOSIS; MITOSIS; and postmitotic functions in differentiated cells. It phosphorylates a number of TRANSCRIPTION FACTORS; and MICROTUBULE-ASSOCIATED PROTEINS.
A cell line derived from cultured tumor cells.
A mitogen-activated protein kinase subfamily that is widely expressed and plays a role in regulation of MEIOSIS; MITOSIS; and post mitotic functions in differentiated cells. The extracellular signal regulated MAP kinases are regulated by a broad variety of CELL SURFACE RECEPTORS and can be activated by certain CARCINOGENS.
Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate.
Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.
Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.
A basic element found in nearly all organized tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes.
An intracellular signaling system involving the MAP kinase cascades (three-membered protein kinase cascades). Various upstream activators, which act in response to extracellular stimuli, trigger the cascades by activating the first member of a cascade, MAP KINASE KINASE KINASES; (MAPKKKs). Activated MAPKKKs phosphorylate MITOGEN-ACTIVATED PROTEIN KINASE KINASES which in turn phosphorylate the MITOGEN-ACTIVATED PROTEIN KINASES; (MAPKs). The MAPKs then act on various downstream targets to affect gene expression. In mammals, there are several distinct MAP kinase pathways including the ERK (extracellular signal-regulated kinase) pathway, the SAPK/JNK (stress-activated protein kinase/c-jun kinase) pathway, and the p38 kinase pathway. There is some sharing of components among the pathways depending on which stimulus originates activation of the cascade.
Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.
The aggregation of soluble ANTIGENS with ANTIBODIES, alone or with antibody binding factors such as ANTI-ANTIBODIES or STAPHYLOCOCCAL PROTEIN A, into complexes large enough to fall out of solution.
The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for VIRUS CULTIVATION and antitumor drug screening assays.
A broad category of carrier proteins that play a role in SIGNAL TRANSDUCTION. They generally contain several modular domains, each of which having its own binding activity, and act by forming complexes with other intracellular-signaling molecules. Signal-transducing adaptor proteins lack enzyme activity, however their activity can be modulated by other signal-transducing enzymes
CELL LINES derived from the CV-1 cell line by transformation with a replication origin defective mutant of SV40 VIRUS, which codes for wild type large T antigen (ANTIGENS, POLYOMAVIRUS TRANSFORMING). They are used for transfection and cloning. (The CV-1 cell line was derived from the kidney of an adult male African green monkey (CERCOPITHECUS AETHIOPS).)
A mitogen-activated protein kinase subfamily that regulates a variety of cellular processes including CELL GROWTH PROCESSES; CELL DIFFERENTIATION; APOPTOSIS; and cellular responses to INFLAMMATION. The P38 MAP kinases are regulated by CYTOKINE RECEPTORS and can be activated in response to bacterial pathogens.
Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein.
Proteins that control the CELL DIVISION CYCLE. This family of proteins includes a wide variety of classes, including CYCLIN-DEPENDENT KINASES, mitogen-activated kinases, CYCLINS, and PHOSPHOPROTEIN PHOSPHATASES as well as their putative substrates such as chromatin-associated proteins, CYTOSKELETAL PROTEINS, and TRANSCRIPTION FACTORS.
Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.
Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.
The smaller subunits of MYOSINS that bind near the head groups of MYOSIN HEAVY CHAINS. The myosin light chains have a molecular weight of about 20 KDa and there are usually one essential and one regulatory pair of light chains associated with each heavy chain. Many myosin light chains that bind calcium are considered "calmodulin-like" proteins.
Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment.
A phorbol ester found in CROTON OIL with very effective tumor promoting activity. It stimulates the synthesis of both DNA and RNA.
A specific inhibitor of phosphoserine/threonine protein phosphatase 1 and 2a. It is also a potent tumor promoter. (Thromb Res 1992;67(4):345-54 & Cancer Res 1993;53(2):239-41)
An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.
Phosphoprotein with protein kinase activity that functions in the G2/M phase transition of the CELL CYCLE. It is the catalytic subunit of the MATURATION-PROMOTING FACTOR and complexes with both CYCLIN A and CYCLIN B in mammalian cells. The maximal activity of cyclin-dependent kinase 1 is achieved when it is fully dephosphorylated.
A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.
An adenine nucleotide containing one phosphate group which is esterified to both the 3'- and 5'-positions of the sugar moiety. It is a second messenger and a key intracellular regulator, functioning as a mediator of activity for a number of hormones, including epinephrine, glucagon, and ACTH.
Transport proteins that carry specific substances in the blood or across cell membranes.
The process of moving proteins from one cellular compartment (including extracellular) to another by various sorting and transport mechanisms such as gated transport, protein translocation, and vesicular transport.
Agents that inhibit PROTEIN KINASES.
Within a eukaryotic cell, a membrane-limited body which contains chromosomes and one or more nucleoli (CELL NUCLEOLUS). The nuclear membrane consists of a double unit-type membrane which is perforated by a number of pores; the outermost membrane is continuous with the ENDOPLASMIC RETICULUM. A cell may contain more than one nucleus. (From Singleton & Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d ed)
One of the mechanisms by which CELL DEATH occurs (compare with NECROSIS and AUTOPHAGOCYTOSIS). Apoptosis is the mechanism responsible for the physiological deletion of cells and appears to be intrinsically programmed. It is characterized by distinctive morphologic changes in the nucleus and cytoplasm, chromatin cleavage at regularly spaced sites, and the endonucleolytic cleavage of genomic DNA; (DNA FRAGMENTATION); at internucleosomal sites. This mode of cell death serves as a balance to mitosis in regulating the size of animal tissues and in mediating pathologic processes associated with tumor growth.
Antibodies directed against immunogen-coupled phosphorylated PEPTIDES corresponding to amino acids surrounding the PHOSPHORYLATION site. They are used to study proteins involved in SIGNAL TRANSDUCTION pathways. (From Methods Mol Biol 2000; 99:177-89)
Elements of limited time intervals, contributing to particular results or situations.
Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.
An enzyme group that specifically dephosphorylates phosphotyrosyl residues in selected proteins. Together with PROTEIN-TYROSINE KINASE, it regulates tyrosine phosphorylation and dephosphorylation in cellular signal transduction and may play a role in cell growth control and carcinogenesis.
A family of non-receptor, PROLINE-rich protein-tyrosine kinases.
A family of ribosomal protein S6 kinases that are structurally distinguished from RIBOSOMAL PROTEIN S6 KINASES, 70-KDA by their apparent molecular size and the fact they contain two functional kinase domains. Although considered RIBOSOMAL PROTEIN S6 KINASES, members of this family are activated via the MAP KINASE SIGNALING SYSTEM and have been shown to act on a diverse array of substrates that are involved in cellular regulation such as RIBOSOMAL PROTEIN S6 and CAMP RESPONSE ELEMENT-BINDING PROTEIN.
Cell lines whose original growing procedure consisted being transferred (T) every 3 days and plated at 300,000 cells per plate (J Cell Biol 17:299-313, 1963). Lines have been developed using several different strains of mice. Tissues are usually fibroblasts derived from mouse embryos but other types and sources have been developed as well. The 3T3 lines are valuable in vitro host systems for oncogenic virus transformation studies, since 3T3 cells possess a high sensitivity to CONTACT INHIBITION.
A large family of signal-transducing adaptor proteins present in wide variety of eukaryotes. They are PHOSPHOSERINE and PHOSPHOTHREONINE binding proteins involved in important cellular processes including SIGNAL TRANSDUCTION; CELL CYCLE control; APOPTOSIS; and cellular stress responses. 14-3-3 proteins function by interacting with other signal-transducing proteins and effecting changes in their enzymatic activity and subcellular localization. The name 14-3-3 derives from numerical designations used in the original fractionation patterns of the proteins.
Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process.
Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics.
Unstable isotopes of phosphorus that decay or disintegrate emitting radiation. P atoms with atomic weights 28-34 except 31 are radioactive phosphorus isotopes.
Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely.
Proteins found in the nucleus of a cell. Do not confuse with NUCLEOPROTEINS which are proteins conjugated with nucleic acids, that are not necessarily present in the nucleus.
Small double-stranded, non-protein coding RNAs (21-31 nucleotides) involved in GENE SILENCING functions, especially RNA INTERFERENCE (RNAi). Endogenously, siRNAs are generated from dsRNAs (RNA, DOUBLE-STRANDED) by the same ribonuclease, Dicer, that generates miRNAs (MICRORNAS). The perfect match of the siRNAs' antisense strand to their target RNAs mediates RNAi by siRNA-guided RNA cleavage. siRNAs fall into different classes including trans-acting siRNA (tasiRNA), repeat-associated RNA (rasiRNA), small-scan RNA (scnRNA), and Piwi protein-interacting RNA (piRNA) and have different specific gene silencing functions.
The complex series of phenomena, occurring between the end of one CELL DIVISION and the end of the next, by which cellular material is duplicated and then divided between two daughter cells. The cell cycle includes INTERPHASE, which includes G0 PHASE; G1 PHASE; S PHASE; and G2 PHASE, and CELL DIVISION PHASE.
Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control (induction or repression) of gene action at the level of transcription or translation.
Paxillin is a signal transducing adaptor protein that localizes to FOCAL ADHESIONS via its four LIM domains. It undergoes PHOSPHORYLATION in response to integrin-mediated CELL ADHESION, and interacts with a variety of proteins including VINCULIN; FOCAL ADHESION KINASE; PROTO-ONCOGENE PROTEIN PP60(C-SRC); and PROTO-ONCOGENE PROTEIN C-CRK.
Oxyvanadium ions in various states of oxidation. They act primarily as ion transport inhibitors due to their inhibition of Na(+)-, K(+)-, and Ca(+)-ATPase transport systems. They also have insulin-like action, positive inotropic action on cardiac ventricular muscle, and other metabolic effects.
A phosphoprotein phosphatase subtype that is comprised of a catalytic subunit and two different regulatory subunits. At least two genes encode isoforms of the protein phosphatase catalytic subunit, while several isoforms of regulatory subunits exist due to the presence of multiple genes and the alternative splicing of their mRNAs. Protein phosphatase 2 acts on a broad variety of cellular proteins and may play a role as a regulator of intracellular signaling processes.
A non-receptor protein tyrosine kinase that is localized to FOCAL ADHESIONS and is a central component of integrin-mediated SIGNAL TRANSDUCTION PATHWAYS. Focal adhesion kinase 1 interacts with PAXILLIN and undergoes PHOSPHORYLATION in response to adhesion of cell surface integrins to the EXTRACELLULAR MATRIX. Phosphorylated p125FAK protein binds to a variety of SH2 DOMAIN and SH3 DOMAIN containing proteins and helps regulate CELL ADHESION and CELL MIGRATION.
A 51-amino acid pancreatic hormone that plays a major role in the regulation of glucose metabolism, directly by suppressing endogenous glucose production (GLYCOGENOLYSIS; GLUCONEOGENESIS) and indirectly by suppressing GLUCAGON secretion and LIPOLYSIS. Native insulin is a globular protein comprised of a zinc-coordinated hexamer. Each insulin monomer containing two chains, A (21 residues) and B (30 residues), linked by two disulfide bonds. Insulin is used as a drug to control insulin-dependent diabetes mellitus (DIABETES MELLITUS, TYPE 1).
A eukayrotic protein serine-threonine phosphatase subtype that dephosphorylates a wide variety of cellular proteins. The enzyme is comprised of a catalytic subunit and regulatory subunit. Several isoforms of the protein phosphatase catalytic subunit exist due to the presence of multiple genes and the alternative splicing of their mRNAs. A large number of proteins have been shown to act as regulatory subunits for this enzyme. Many of the regulatory subunits have additional cellular functions.
The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.
A strain of albino rat used widely for experimental purposes because of its calmness and ease of handling. It was developed by the Sprague-Dawley Animal Company.
Diffusible gene products that act on homologous or heterologous molecules of viral or cellular DNA to regulate the expression of proteins.
The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties.
The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.
The sum of the weight of all the atoms in a molecule.
A type of CELL NUCLEUS division by means of which the two daughter nuclei normally receive identical complements of the number of CHROMOSOMES of the somatic cells of the species.
A serine-threonine protein kinase family whose members are components in protein kinase cascades activated by diverse stimuli. These MAPK kinases phosphorylate MITOGEN-ACTIVATED PROTEIN KINASES and are themselves phosphorylated by MAP KINASE KINASE KINASES. JNK kinases (also known as SAPK kinases) are a subfamily.
The relationship between the dose of an administered drug and the response of the organism to the drug.
A cell line generated from human embryonic kidney cells that were transformed with human adenovirus type 5.
A group of protein-serine-threonine kinases that was originally identified as being responsible for the PHOSPHORYLATION of CASEINS. They are ubiquitous enzymes that have a preference for acidic proteins. Casein kinases play a role in SIGNAL TRANSDUCTION by phosphorylating a variety of regulatory cytoplasmic and regulatory nuclear proteins.
Commonly observed structural components of proteins formed by simple combinations of adjacent secondary structures. A commonly observed structure may be composed of a CONSERVED SEQUENCE which can be represented by a CONSENSUS SEQUENCE.
Connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules.
A 6-kDa polypeptide growth factor initially discovered in mouse submaxillary glands. Human epidermal growth factor was originally isolated from urine based on its ability to inhibit gastric secretion and called urogastrone. Epidermal growth factor exerts a wide variety of biological effects including the promotion of proliferation and differentiation of mesenchymal and EPITHELIAL CELLS. It is synthesized as a transmembrane protein which can be cleaved to release a soluble active form.
A family of protein serine/threonine kinases which act as intracellular signalling intermediates. Ribosomal protein S6 kinases are activated through phosphorylation in response to a variety of HORMONES and INTERCELLULAR SIGNALING PEPTIDES AND PROTEINS. Phosphorylation of RIBOSOMAL PROTEIN S6 by enzymes in this class results in increased expression of 5' top MRNAs. Although specific for RIBOSOMAL PROTEIN S6 members of this class of kinases can act on a number of substrates within the cell. The immunosuppressant SIROLIMUS inhibits the activation of ribosomal protein S6 kinases.
Inorganic salts of phosphoric acid.
A structurally-related group of signaling proteins that are phosphorylated by the INSULIN RECEPTOR PROTEIN-TYROSINE KINASE. The proteins share in common an N-terminal PHOSPHOLIPID-binding domain, a phosphotyrosine-binding domain that interacts with the phosphorylated INSULIN RECEPTOR, and a C-terminal TYROSINE-rich domain. Upon tyrosine phosphorylation insulin receptor substrate proteins interact with specific SH2 DOMAIN-containing proteins that are involved in insulin receptor signaling.
Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.
Protein kinases that control cell cycle progression in all eukaryotes and require physical association with CYCLINS to achieve full enzymatic activity. Cyclin-dependent kinases are regulated by phosphorylation and dephosphorylation events.
Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor.
The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.
A subgroup of mitogen-activated protein kinases that activate TRANSCRIPTION FACTOR AP-1 via the phosphorylation of C-JUN PROTEINS. They are components of intracellular signaling pathways that regulate CELL PROLIFERATION; APOPTOSIS; and CELL DIFFERENTIATION.
A serine threonine kinase that controls a wide range of growth-related cellular processes. The protein is referred to as the target of RAPAMYCIN due to the discovery that SIROLIMUS (commonly known as rapamycin) forms an inhibitory complex with TACROLIMUS BINDING PROTEIN 1A that blocks the action of its enzymatic activity.
A ribosomal protein that may play a role in controlling cell growth and proliferation. It is a major substrate of RIBOSOMAL PROTEIN S6 KINASES and plays a role in regulating the translation (TRANSLATION, GENETIC) of RNAs that contain an RNA 5' TERMINAL OLIGOPYRIMIDINE SEQUENCE.
A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS.
All of the processes involved in increasing CELL NUMBER including CELL DIVISION.
Filamentous proteins that are the main constituent of the thin filaments of muscle fibers. The filaments (known also as filamentous or F-actin) can be dissociated into their globular subunits; each subunit is composed of a single polypeptide 375 amino acids long. This is known as globular or G-actin. In conjunction with MYOSINS, actin is responsible for the contraction and relaxation of muscle.
Major constituent of the cytoskeleton found in the cytoplasm of eukaryotic cells. They form a flexible framework for the cell, provide attachment points for organelles and formed bodies, and make communication between parts of the cell possible.
A signal transducer and activator of transcription that mediates cellular responses to INTERLEUKIN-6 family members. STAT3 is constitutively activated in a variety of TUMORS and is a major downstream transducer for the CYTOKINE RECEPTOR GP130.
The part of a cell that contains the CYTOSOL and small structures excluding the CELL NUCLEUS; MITOCHONDRIA; and large VACUOLES. (Glick, Glossary of Biochemistry and Molecular Biology, 1990)
A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey (C. pygerythrus) is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research.
A rather large group of enzymes comprising not only those transferring phosphate but also diphosphate, nucleotidyl residues, and others. These have also been subdivided according to the acceptor group. (From Enzyme Nomenclature, 1992) EC 2.7.
A serine-threonine kinase that plays important roles in CELL DIFFERENTIATION; CELL MIGRATION; and CELL DEATH of NERVE CELLS. It is closely related to other CYCLIN-DEPENDENT KINASES but does not seem to participate in CELL CYCLE regulation.
A casein kinase that was originally described as a monomeric enzyme with a molecular weight of 30-40 kDa. Several ISOENZYMES of casein kinase I have been found which are encoded by separate genes. Many of the casein kinase I isoenzymes have been shown to play distinctive roles in intracellular SIGNAL TRANSDUCTION.
Regions of AMINO ACID SEQUENCE similarity in the SRC-FAMILY TYROSINE KINASES that fold into specific functional tertiary structures. The SH1 domain is a CATALYTIC DOMAIN. SH2 and SH3 domains are protein interaction domains. SH2 usually binds PHOSPHOTYROSINE-containing proteins and SH3 interacts with CYTOSKELETAL PROTEINS.
RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.
A negative regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (GENE EXPRESSION REGULATION), mRNAs (RNA, MESSENGER), and proteins.
The degree of similarity between sequences of amino acids. This information is useful for the analyzing genetic relatedness of proteins and species.
Membrane-associated tyrosine-specific kinases encoded by the c-src genes. They have an important role in cellular growth control. Truncation of carboxy-terminal residues in pp60(c-src) leads to PP60(V-SRC) which has the ability to transform cells. This kinase pp60 c-src should not be confused with csk, also known as c-src kinase.
The fission of a CELL. It includes CYTOKINESIS, when the CYTOPLASM of a cell is divided, and CELL NUCLEUS DIVISION.
An analytical method used in determining the identity of a chemical based on its mass using mass analyzers/mass spectrometers.
A cell surface receptor involved in regulation of cell growth and differentiation. It is specific for EPIDERMAL GROWTH FACTOR and EGF-related peptides including TRANSFORMING GROWTH FACTOR ALPHA; AMPHIREGULIN; and HEPARIN-BINDING EGF-LIKE GROWTH FACTOR. The binding of ligand to the receptor causes activation of its intrinsic tyrosine kinase activity and rapid internalization of the receptor-ligand complex into the cell.
The species Oryctolagus cuniculus, in the family Leporidae, order LAGOMORPHA. Rabbits are born in burrows, furless, and with eyes and ears closed. In contrast with HARES, rabbits have 22 chromosome pairs.
A group of phenyl benzopyrans named for having structures like FLAVONES.
Small chromosomal proteins (approx 12-20 kD) possessing an open, unfolded structure and attached to the DNA in cell nuclei by ionic linkages. Classification into the various types (designated histone I, histone II, etc.) is based on the relative amounts of arginine and lysine in each.
A group of hydrolases which catalyze the hydrolysis of monophosphoric esters with the production of one mole of orthophosphate. EC 3.1.3.
Strains of mice in which certain GENES of their GENOMES have been disrupted, or "knocked-out". To produce knockouts, using RECOMBINANT DNA technology, the normal DNA sequence of the gene being studied is altered to prevent synthesis of a normal gene product. Cloned cells in which this DNA alteration is successful are then injected into mouse EMBRYOS to produce chimeric mice. The chimeric mice are then bred to yield a strain in which all the cells of the mouse contain the disrupted gene. Knockout mice are used as EXPERIMENTAL ANIMAL MODELS for diseases (DISEASE MODELS, ANIMAL) and to clarify the functions of the genes.
Derivatives of the steroid androstane having two double bonds at any site in any of the rings.
Eukaryotic initiation factor of protein synthesis. In higher eukaryotes the factor consists of three subunits: alpha, beta, and gamma. As initiation proceeds, eIF-2 forms a ternary complex with Met-tRNAi and GTP.
Intracellular signaling protein kinases that play a signaling role in the regulation of cellular energy metabolism. Their activity largely depends upon the concentration of cellular AMP which is increased under conditions of low energy or metabolic stress. AMP-activated protein kinases modify enzymes involved in LIPID METABOLISM, which in turn provide substrates needed to convert AMP into ATP.
CELL LINE derived from the ovary of the Chinese hamster, Cricetulus griseus (CRICETULUS). The species is a favorite for cytogenetic studies because of its small chromosome number. The cell line has provided model systems for the study of genetic alterations in cultured mammalian cells.
A cell surface receptor for INSULIN. It comprises a tetramer of two alpha and two beta subunits which are derived from cleavage of a single precursor protein. The receptor contains an intrinsic TYROSINE KINASE domain that is located within the beta subunit. Activation of the receptor by INSULIN results in numerous metabolic changes including increased uptake of GLUCOSE into the liver, muscle, and ADIPOSE TISSUE.
Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components.
Processes that stimulate the GENETIC TRANSCRIPTION of a gene or set of genes.
The span of viability of a cell characterized by the capacity to perform certain functions such as metabolism, growth, reproduction, some form of responsiveness, and adaptability.
A ubiquitously expressed protein kinase that is involved in a variety of cellular SIGNAL PATHWAYS. Its activity is regulated by a variety of signaling protein tyrosine kinase.
A protein that has been shown to function as a calcium-regulated transcription factor as well as a substrate for depolarization-activated CALCIUM-CALMODULIN-DEPENDENT PROTEIN KINASES. This protein functions to integrate both calcium and cAMP signals.
Ubiquitous, inducible, nuclear transcriptional activator that binds to enhancer elements in many different cell types and is activated by pathogenic stimuli. The NF-kappa B complex is a heterodimer composed of two DNA-binding subunits: NF-kappa B1 and relA.
Electrophoresis in which a second perpendicular electrophoretic transport is performed on the separate components resulting from the first electrophoresis. This technique is usually performed on polyacrylamide gels.
A family of ribosomal protein S6 kinases that are considered the major physiological kinases for RIBOSOMAL PROTEIN S6. Unlike RIBOSOMAL PROTEIN S6 KINASES, 90KDa the proteins in this family are sensitive to the inhibitory effects of RAPAMYCIN and contain a single kinase domain. They are referred to as 70kDa proteins, however ALTERNATIVE SPLICING of mRNAs for proteins in this class also results in 85kDa variants being formed.
A multifunctional calcium-calmodulin-dependent protein kinase subtype that occurs as an oligomeric protein comprised of twelve subunits. It differs from other enzyme subtypes in that it lacks a phosphorylatable activation domain that can respond to CALCIUM-CALMODULIN-DEPENDENT PROTEIN KINASE KINASE.
Src-family kinases that associate with T-CELL ANTIGEN RECEPTOR and phosphorylate a wide variety of intracellular signaling molecules.
A phosphoinositide phospholipase C subtype that is primarily regulated by PROTEIN-TYROSINE KINASES. It is structurally related to PHOSPHOLIPASE C DELTA with the addition of SRC HOMOLOGY DOMAINS and pleckstrin homology domains located between two halves of the CATALYTIC DOMAIN.
Microtubule-associated proteins that are mainly expressed in neurons. Tau proteins constitute several isoforms and play an important role in the assembly of tubulin monomers into microtubules and in maintaining the cytoskeleton and axonal transport. Aggregation of specific sets of tau proteins in filamentous inclusions is the common feature of intraneuronal and glial fibrillar lesions (NEUROFIBRILLARY TANGLES; NEUROPIL THREADS) in numerous neurodegenerative disorders (ALZHEIMER DISEASE; TAUOPATHIES).
Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are linear polypeptides that are normally synthesized on RIBOSOMES.
An enzyme that phosphorylates myosin light chains in the presence of ATP to yield myosin-light chain phosphate and ADP, and requires calcium and CALMODULIN. The 20-kDa light chain is phosphorylated more rapidly than any other acceptor, but light chains from other myosins and myosin itself can act as acceptors. The enzyme plays a central role in the regulation of smooth muscle contraction.
A primary source of energy for living organisms. It is naturally occurring and is found in fruits and other parts of plants in its free state. It is used therapeutically in fluid and nutrient replacement.
A gene silencing phenomenon whereby specific dsRNAs (RNA, DOUBLE-STRANDED) trigger the degradation of homologous mRNA (RNA, MESSENGER). The specific dsRNAs are processed into SMALL INTERFERING RNA (siRNA) which serves as a guide for cleavage of the homologous mRNA in the RNA-INDUCED SILENCING COMPLEX. DNA METHYLATION may also be triggered during this process.
Gated transport mechanisms by which proteins or RNA are moved across the NUCLEAR MEMBRANE.
A subclass of phospholipases that hydrolyze the phosphoester bond found in the third position of GLYCEROPHOSPHOLIPIDS. Although the singular term phospholipase C specifically refers to an enzyme that catalyzes the hydrolysis of PHOSPHATIDYLCHOLINE (EC, it is commonly used in the literature to refer to broad variety of enzymes that specifically catalyze the hydrolysis of PHOSPHATIDYLINOSITOLS.
The network of filaments, tubules, and interconnecting filamentous bridges which give shape, structure, and organization to the cytoplasm.
The movement of cells from one location to another. Distinguish from CYTOKINESIS which is the process of dividing the CYTOPLASM of a cell.
Different forms of a protein that may be produced from different GENES, or from the same gene by ALTERNATIVE SPLICING.
Proteins that are normally involved in holding cellular growth in check. Deficiencies or abnormalities in these proteins may lead to unregulated cell growth and tumor development.
A group of enzymes that transfers a phosphate group onto an alcohol group acceptor. EC 2.7.1.
An indolocarbazole that is a potent PROTEIN KINASE C inhibitor which enhances cAMP-mediated responses in human neuroblastoma cells. (Biochem Biophys Res Commun 1995;214(3):1114-20)
Amino acids that contain phosphorus as an integral part of the molecule.
Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.
A cytoplasmic serine threonine kinase involved in regulating CELL DIFFERENTIATION and CELLULAR PROLIFERATION. Overexpression of this enzyme has been shown to promote PHOSPHORYLATION of BCL-2 PROTO-ONCOGENE PROTEINS and chemoresistance in human acute leukemia cells.
A Janus kinase subtype that is involved in signaling from GROWTH HORMONE RECEPTORS; PROLACTIN RECEPTORS; and a variety of CYTOKINE RECEPTORS such as ERYTHROPOIETIN RECEPTORS and INTERLEUKIN RECEPTORS. Dysregulation of Janus kinase 2 due to GENETIC TRANSLOCATIONS have been associated with a variety of MYELOPROLIFERATIVE DISORDERS.
A heat-stable, low-molecular-weight activator protein found mainly in the brain and heart. The binding of calcium ions to this protein allows this protein to bind to cyclic nucleotide phosphodiesterases and to adenyl cyclase with subsequent activation. Thereby this protein modulates cyclic AMP and cyclic GMP levels.
A group of intracellular-signaling serine threonine kinases that bind to RHO GTP-BINDING PROTEINS. They were originally found to mediate the effects of rhoA GTP-BINDING PROTEIN on the formation of STRESS FIBERS and FOCAL ADHESIONS. Rho-associated kinases have specificity for a variety of substrates including MYOSIN-LIGHT-CHAIN PHOSPHATASE and LIM KINASES.
Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye.
Compounds of the general formula R-O-R arranged in a ring or crown formation.
Five-membered heterocyclic ring structures containing an oxygen in the 1-position and a nitrogen in the 3-position, in distinction from ISOXAZOLES where they are at the 1,2 positions.
A group of PROTEIN-SERINE-THREONINE KINASES which activate critical signaling cascades in double strand breaks, APOPTOSIS, and GENOTOXIC STRESS such as ionizing ultraviolet A light, thereby acting as a DNA damage sensor. These proteins play a role in a wide range of signaling mechanisms in cell cycle control.
Adherence of cells to surfaces or to other cells.
Four carbon unsaturated hydrocarbons containing two double bonds.
An isoflavonoid derived from soy products. It inhibits PROTEIN-TYROSINE KINASE and topoisomerase-II (DNA TOPOISOMERASES, TYPE II); activity and is used as an antineoplastic and antitumor agent. Experimentally, it has been shown to induce G2 PHASE arrest in human and murine cell lines and inhibits PROTEIN-TYROSINE KINASE.
Monomeric subunits of primarily globular ACTIN and found in the cytoplasmic matrix of almost all cells. They are often associated with microtubules and may play a role in cytoskeletal function and/or mediate movement of the cell or the organelles within the cell.
An abundant 43-kDa mitogen-activated protein kinase kinase subtype with specificity for MITOGEN-ACTIVATED PROTEIN KINASE 1 and MITOGEN-ACTIVATED PROTEIN KINASE 3.
A phosphoprotein phosphatase that is specific for MYOSIN LIGHT CHAINS. It is composed of three subunits, which include a catalytic subunit, a myosin binding subunit, and a third subunit of unknown function.
Compounds with a six membered aromatic ring containing NITROGEN. The saturated version is PIPERIDINES.
A signal transducer and activator of transcription that mediates cellular responses to INTERFERONS. Stat1 interacts with P53 TUMOR SUPPRESSOR PROTEIN and regulates expression of GENES involved in growth control and APOPTOSIS.
A ubiquitously expressed raf kinase subclass that plays an important role in SIGNAL TRANSDUCTION. The c-raf Kinases are MAP kinase kinase kinases that have specificity for MAP KINASE KINASE 1 and MAP KINASE KINASE 2.
A transferase that catalyzes the addition of aliphatic, aromatic, or heterocyclic FREE RADICALS as well as EPOXIDES and arene oxides to GLUTATHIONE. Addition takes place at the SULFUR. It also catalyzes the reduction of polyol nitrate by glutathione to polyol and nitrite.
A peptide initiation factor that binds specifically to the 5' MRNA CAP STRUCTURE of MRNA in the CYTOPLASM. It is a component of the trimeric complex EIF4F.
Injuries to DNA that introduce deviations from its normal, intact structure and which may, if left unrepaired, result in a MUTATION or a block of DNA REPLICATION. These deviations may be caused by physical or chemical agents and occur by natural or unnatural, introduced circumstances. They include the introduction of illegitimate bases during replication or by deamination or other modification of bases; the loss of a base from the DNA backbone leaving an abasic site; single-strand breaks; double strand breaks; and intrastrand (PYRIMIDINE DIMERS) or interstrand crosslinking. Damage can often be repaired (DNA REPAIR). If the damage is extensive, it can induce APOPTOSIS.
The muscle tissue of the HEART. It is composed of striated, involuntary muscle cells (MYOCYTES, CARDIAC) connected to form the contractile pump to generate blood flow.
A continuous cell line of high contact-inhibition established from NIH Swiss mouse embryo cultures. The cells are useful for DNA transfection and transformation studies. (From ATCC [Internet]. Virginia: American Type Culture Collection; c2002 [cited 2002 Sept 26]. Available from http://www.atcc.org/)
A subtype of striated muscle, attached by TENDONS to the SKELETON. Skeletal muscles are innervated and their movement can be consciously controlled. They are also called voluntary muscles.

Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own. (1/72374)

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners.  (+info)

Cell polarization: chemotaxis gets CRACKing. (2/72374)

An early stage in the establishment of cell polarity during chemotaxis of Dictyostelium dicoideum has been identified by a recent study; the new results also show that the development of cell polarity does not rely upon cytoskeletal rearrangement, and may use a spatial sensing mechanism.  (+info)

The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors. (3/72374)

BACKGROUND: The adaptor protein Gads is a Grb2-related protein originally identified on the basis of its interaction with the tyrosine-phosphorylated form of the docking protein Shc. Gads protein expression is restricted to hematopoietic tissues and cell lines. Gads contains a Src homology 2 (SH2) domain, which has previously been shown to have a similar binding specificity to that of Grb2. Gads also possesses two SH3 domains, but these have a distinct binding specificity to those of Grb2, as Gads does not bind to known Grb2 SH3 domain targets. Here, we investigated whether Gads is involved in T-cell signaling. RESULTS: We found that Gads is highly expressed in T cells and that the SLP-76 adaptor protein is a major Gads-associated protein in vivo. The constitutive interaction between Gads and SLP-76 was mediated by the carboxy-terminal SH3 domain of Gads and a 20 amino-acid proline-rich region in SLP-76. Gads also coimmunoprecipitated the tyrosine-phosphorylated form of the linker for activated T cells (LAT) adaptor protein following cross-linking of the T-cell receptor; this interaction was mediated by the Gads SH2 domain. Overexpression of Gads and SLP-76 resulted in a synergistic augmentation of T-cell signaling, as measured by activation of nuclear factor of activated T cells (NFAT), and this cooperation required a functional Gads SH2 domain. CONCLUSIONS: These results demonstrate that Gads plays an important role in T-cell signaling via its association with SLP-76 and LAT. Gads may promote cross-talk between the LAT and SLP-76 signaling complexes, thereby coupling membrane-proximal events to downstream signaling pathways.  (+info)

Tyrosine phosphorylation is required for actin-based motility of vaccinia but not Listeria or Shigella. (4/72374)

Studies of the actin-based motility of pathogens have provided important insights into the events occurring at the leading edge of motile cells [1] [2] [3]. To date, several actin-cytoskeleton-associated proteins have been implicated in the motility of Listeria or Shigella: vasodilator-stimulated phosphoprotein (VASP), vinculin and the actin-related protein complex of Arp2 and Arp3 [4] [5] [6] [7]. To further investigate the underlying mechanism of actin-tail assembly, we examined the localization of components of the actin cytoskeleton including Arp3, VASP, vinculin and zyxin during vaccinia, Listeria and Shigella infections. The most striking difference between the systems was that a phosphotyrosine signal was observed only at the site of vaccinia actin-tail assembly. Micro-injection experiments demonstrated that a phosphotyrosine protein plays an important role in vaccinia actin-tail formation. In addition, we observed a phosphotyrosine signal on clathrin-coated vesicles that have associated actin-tail-like structures and on endogenous vesicles in Xenopus egg extracts which are able to nucleate actin tails [8] [9]. Our observations indicate that a host phosphotyrosine protein is required for the nucleation of actin filaments by vaccinia and suggest that this phosphoprotein might be associated with cellular membranes that can nucleate actin.  (+info)

Intracellular signalling: PDK1--a kinase at the hub of things. (5/72374)

Phosphoinositide-dependent kinase 1 (PDK1) is at the hub of many signalling pathways, activating PKB and PKC isoenzymes, as well as p70 S6 kinase and perhaps PKA. PDK1 action is determined by colocalization with substrate and by target site availability, features that may enable it to operate in both resting and stimulated cells.  (+info)

Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci. (6/72374)

BACKGROUND: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents. RESULTS: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2"). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C. CONCLUSIONS: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design.  (+info)

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications. (7/72374)

BACKGROUND: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. RESULTS: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. CONCLUSIONS: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.  (+info)

Tyrosine phosphorylation and complex formation of Cbl-b upon T cell receptor stimulation. (8/72374)

Cbl-b, a mammalian homolog of Cbl, consists of an N-terminal region (Cbl-b-N) highly homologous to oncogenic v-Cbl, a Ring finger, and a C-terminal region containing multiple proline-rich stretches and potential tyrosine phosphorylation sites. In the present study, we demonstrate that upon engagement of the T cell receptor (TCR), endogenous Cbl-b becomes rapidly tyrosine-phosphorylated. In heterogeneous COS-1 cells, Cbl-b was phosphorylated on tyrosine residues by both Syk- (Syk/Zap-70) and Src- (Fyn/Lck) family kinases, with Syk kinase inducing the most prominent effect. Syk associates and phosphorylates Cbl-b in Jurkat T cells. A Tyr-316 Cbl-binding site in Syk was required for the association with and for the maximal tyrosine phosphorylation of Cbl-b. Mutation at a loss-of-function site (Gly-298) in Cbl-b-N disrupts its interaction with Syk. Cbl-b constitutively binds Grb2 and becomes associated with Crk-L upon TCR stimulation. The Grb2- and the Crk-L-binding regions were mapped to the C-terminus of Cbl-b. The Crk-L-binding sites were further determined to be Y655DVP and Y709KIP, with the latter being the primary binding site. Taken together, these results implicate that Cbl-b is involved in TCR-mediated intracellular signaling pathways.  (+info)

TY - JOUR. T1 - Molecular Basis for the Substrate Specificity of a Serine/Threonine-Specific Protein Kinase. AU - Lee, Tae Ryong. AU - Mendelow, Marianne. AU - Srinivasan, Jaya. AU - Kwon, Young Guen. AU - Lawrence, David S.. PY - 1993/11/1. Y1 - 1993/11/1. N2 - Protein kinases typically phosphorylate the aliphatic alcohols of serine/threonine residues or the aromatic alcohol of tyrosine residues but not both. We report herein the first example of aromatic alcohol phosphorylation by a serine/threonine-specific protein kinase. The cAMP-dependent protein kinase phosphorylates the C-terminal aromatic alcohol in the active site-directed peptide Gly-Arg-Thr-Gly-Arg-Arg-Asn-(o-aminophenol). In contrast, corresponding peptides containing m- and p-aminophenols failed to serve as protein kinase substrates. These results indicate that the orientation of the aromatic hydroxyl group relative to the adjacent peptide backbone bond is a critical structural motif employed in substrate recognition by the enzyme. ...
(2003) Mandell. American Journal of Pathology. Until recently, the investigation of protein phosphorylation was limited to biochemical studies of enzyme activities in homogenized tissues. The availability of hundreds of phosphorylation state-specific antibodies (PSSAs) now makes possible the stud...
Summary: Error-free cell division depends on spatial and temporal cues regulating micron-scale organization. In current models, substrate phosphorylation plays a central role in generating these regulatory cues. While the kinase and phosphatase localizations during cell division have been extensively analyzed, the dynamics of phosphorylation remain poorly characterized. To fill this gap in our knowledge, we developed FRET-based sensors to examine in dividing cells the substrate phosphorylation dynamics that depend on Aurora kinase, a conserved cell cycle regulator in eukaryotes and an anti-cancer drug target. Quantitative analysis of phosphorylation dynamics, using sensors targeted to chromosomes or centromeres, revealed that as chromosomes segregate substrate phosphorylation levels depend more on intracellular position than on time elapsed after anaphase. These data, along with immunofluorescence analyses using phosphorylation-specific antibodies, revealed that a spatial phosphorylation ...
Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and Stat5b proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether Stat5a and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1-5 min of IL2 stimulation, whereas ...
PKA phosphorylation increases the tyrosine kinase activity of Csk towards an endogenous substrate. Tyrosine phosphorylation of heat-inactivated (65°C for 10 mi
Post-translational modification (PTM) of proteins regulates many biological phenomena [1]. Among the several kinds of PTM, phosphorylation affects enzymatic activity, conformations, interactions, degradation, and localization of proteins, among other effects [2-4]; one of the critical roles of phosphorylation is in the control of protein signaling [5]. More than 500 protein kinases are thought to regulate protein signaling in humans [6]. In protein signaling, various reaction cascades transmit and amplify signals in a highly regulated manner by means of reversible site-specific protein phosphorylation [5]. Kinases recognize the specific surrounding sequences of phosphosites when they phosphorylate their targets, and the majority of the identified kinases are thought to have their own unique target sequences, which are known as motif sequences [7].. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), combined with phosphopeptide enrichment technology [8], is a powerful ...
In vitro phosphorylation of the regulatory subunit of yeast cAMP-dependent protein kinase was studied. The cAMP-binding regulatory subunit (R subunit) can be multiply phosphorylated. Three distinct phosphorylation sites were inferred from the different ATP concentrations required for phosphorylation and from the presence of two discrete mobility shifts in NaDodSO4/polyacrylamide gel electrophoresis of the R subunit on phosphorylation. Limited tryptic digestion of the phosphorylated R subunit showed that a Mr 37,000 cAMP-binding peptide contained one of the phosphorylation sites and that a separate Mr 12,000 peptide contained another phosphorylation site. The yeast R subunit is therefore similar to the type II R subunit of mammalian origin, although it has a larger Mr (64,000 vs. 58,000) and is multiply phosphorylated. In vivo, both phosphorylated and unphosphorylated forms of the R subunit were found in cells grown in lactate or to stationary phase in 1.5% glucose, while cells grown in 5% ...
Cardiac Na+/K+ ATPase plays a pivotal role in maintaining the Na+ transmembrane gradient which is essential for normal cardiac function. Elevation of intracellular Na+ ([Na+]i) is a key contributor to contractile and electrical dysfunction in a variety of pathologies. Phospholemman (PLM), is the cardiac specific member of the FXYD family of small membrane spanning proteins, and forms a complex with Na+/K+ ATPase pump. PLM regulates the pump by exerting a tonic inhibition which is relieved by PKA or PKC phosphorylation at 3 serine (Ser)/threonine (Thr) residues in its cytoplasmic tail (Ser63, Ser68, Thr/Ser69). Phosphorylation at any of these sites results in disinhibition of the pump and even active stimulation. The work described in this thesis investigates the role of PLM in regulating the Na+/K+ ATPase and the subsequent effect on [Na+]i. A new mouse model, PLM3SA, has been created by mutating all 3 Ser residues to alanine (Ala) rendering the protein unphosphorylatable. This inability to ...
TY - JOUR. T1 - Monocytes, but not T cells, respond to insulin with Akt(S473) phosphorylation independent of the donor glucometabolic state.. AU - Thewissen, M.M.. AU - van de Gaar, J.. AU - den Boer, A.T.. AU - Marjet Munsters, M.J.M.. AU - Blaak, E.E.. AU - Duijvestijn, A.M.. PY - 2014/1/1. Y1 - 2014/1/1. N2 - BACKGROUND: Obesity is associated with insulin resistance and chronic low-grade inflammation. Insulin has been described to have anti-inflammatory effects in immune cells. Therefore, insulin resistance in immune cells can be expected to have important consequences for immune function. Here, we investigate whether freshly isolated monocytes and T cells, isolated from study subjects with a normal or disturbed glucometabolic state, respond to insulin with phosphorylation of Akt, a key molecule in the insulin signalling pathway. METHODS: 25 study subjects were enrolled in the study. An oral glucose tolerance test (OGTT) was performed and, from fasting insulin and glucose, HOMA-IR was ...
Protein phosphorylation regulates a large variety of biological processes in all living cells. In pathogenic bacteria, the study of serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation has shed light on the course of infectious diseases, from adherence to host cells to pathogen virulence, replication, and persistence. Mass spectrometry (MS)-based phosphoproteomics has provided global maps of Ser/Thr/Tyr phosphosites in bacterial pathogens. Despite recent developments, a quantitative and dynamic view of phosphorylation events that occur during bacterial pathogenesis is currently lacking. Temporal, spatial, and subpopulation resolution of phosphorylation data is required to identify key regulatory nodes underlying bacterial pathogenesis. Herein, we discuss how technological improvements in sample handling, MS instrumentation, data processing, and machine learning should improve bacterial phosphoproteomic datasets and the information extracted from them. Such information is expected to
This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases., 该方案描述了人蛋白质c-Myc的体外去磷酸化测定的实验程序。 该方案可适用于检测其他Ser / Thr磷酸酶的磷酸酶活性。【背景】羧基末端结构域RNA聚合酶II多肽小型磷酸酶1(SCP1,也称为CTDSP1或NLI-IF)属于FCP / SCP磷酸酶家族,最初被报道使RNA聚合酶II的C-末端结构域(CTD)脱磷酸化 (Yeo等,2003)。 Smad2,3(Wrighton等,2006),Snail(Wu等,2009),PML(Lin等,2014)和c-Myc(Wang等,2016)也被鉴定为底物 的SCP1。
TY - JOUR. T1 - Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells. AU - Chen, Yunhao. AU - Low, Teck Yew. AU - Choong, Lee Yee. AU - Ray, Rajarshi Sankar. AU - Tan, Yee Ling. AU - Toy, Weiyi. AU - Lin, Qingsong. AU - Boon, Keong Ang. AU - Chee, Hong Wong. AU - Lim, Simin. AU - Li, Bin. AU - Hew, Choy Leong. AU - Sze, Newman Siu Kwan. AU - Druker, Brian. AU - Lim, Yoon Pin. PY - 2007/7. Y1 - 2007/7. N2 - With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be ...
TY - JOUR. T1 - Optimizing an intermittent stretch paradigm using ERK1/2 phosphorylation results in increased collagen synthesis in engineered ligaments. AU - Paxton, Jennifer Z.. AU - Hagerty, Paul. AU - Andrick, Jonathan J.. AU - Baar, Keith. PY - 2012/2/1. Y1 - 2012/2/1. N2 - Dynamic mechanical input is believed to play a critical role in the development of functional musculoskeletal tissues. To study this phenomenon, cyclic uniaxial mechanical stretch was applied to engineered ligaments using a custom-built bioreactor and the effects of different stretch frequency, amplitude, and duration were determined. Stretch acutely increased the phosphorylation of p38 (3.5±0.74-fold), S6K1 (3.9±0.19-fold), and ERK1/2 (2.45±0.32-fold). The phosphorylation of ERK1/2 was dependent on time, rather than on frequency or amplitude, within these constructs. ERK1/2 phosphorylation was similar following stretch at frequencies from 0.1 to 1 Hz and amplitudes from 2.5% to 15%, whereas phosphorylation reached ...
Oscillatory protein phosphorylation regulates the major phase transitions of the cell division cycle. The overall amount of phosphorylation is especially high during mitosis (37, 38), and several large-scale studies have identified sets of phosphorylation sites present during mitosis (8, 11-13, 39). These studies, although mostly performed on a phosphoproteome scale, are still far from complete due to the complexity and variance in protein abundance within the proteome (40). Because phosphoproteomic studies usually rely on phosphopeptide enrichment, the information about the unphosphorylated proteins is lost, and it thus remains difficult to estimate the protein coverage in these studies. Here, we have taken a complementary approach to analyze mitotic phosphorylation within purified mitotic protein complexes. The much lower sample complexity allowed simultaneous analysis of phosphorylated and unphosphorylated peptides to obtain a measure of sequence coverage for each analyzed protein and ...
In eukaryotes, hundreds of protein kinases (PKs) specifically and precisely modify thousands of substrates at specific amino acid residues to faithfully orchestrate numerous biological processes, and reversibly determine the cellular dynamics and plasticity. Although over 100,000 phosphorylation sites (p-sites) have been experimentally identified from phosphoproteomic studies, the regulatory PKs for most of these sites still remain to be characterized. Here, we present a novel software package of iGPS for the prediction of in vivo site-specific kinase-substrate relations mainly from the phosphoproteomic data. By critical evaluations and comparisons, the performance of iGPS is satisfying and better than other existed tools. Based on the prediction results, we modeled protein phosphorylation networks and observed that the eukaryotic phospho-regulation is poorly conserved at the site and substrate levels. With an integrative procedure, we conducted a large-scale phosphorylation analysis of human ...
β-Catenin phosphorylation plays important roles in modulating its functions, but the effects of different phosphorylated forms of β-catenin in response to heterocellular interaction are unclear. Here we investigated whether distinct modes of phosphorylation on β-catenin could be triggered through heterocellular interactions between endothelial cells (ECs) and smooth muscle cells (SMCs), and the consequent modulation of EC functions. ECs were cocultured with SMCs to initiate direct contact and paracrine interaction. EC-SMC coculture induced EC β-catenin phosphorylations simultaneously at tyrosine 142 (Tyr142) and serine 45/threonine 41 (Ser45/Thr41) at the cytoplasm/nuclei and the membrane, respectively. Treating ECs with SMC-conditional medium induced β-catenin phosphorylation only at Ser45/Thr41. These findings indicate that different phosphorylation effects of EC-SMC coculture were induced through heterocellular direct contact and paracrine effects, respectively. Using specific blocking ...
FLCN interacts with AMPK via FNIP1 and/or FNIP2, and regulates mTOR signalling (Baba et al., 2006; Hasumi et al., 2008, Takagi et al., 2008). Phosphorylation at S62 of FLCN increases the FLCN-FNIP complexs affinity for AMPK, while phosphorylation at S302 decreases FLCNs affinity for AMPK (Piao et al., 2009, Wang et al., 2010). AMPK is an important energy sensing protein, which inhibits anabolic growth via mTOR signalling and stimulates autophagy to promote cell survival when energy supply is low (Alers et al., 2012).. Loss of FLCN in mouse embryonic fibroblasts (MEFs), leads to the constitutive activation of AMPK (Yan et al., 2014). This ultimately activates HIF signalling and leads to metabolic changes consistent with the Warburg Effect within FLCN-null cells. A nonphosphorylatable FLCN S62A mutant was unable to bind and inhibit AMPK, meaning that FLCN-FNIP binding to AMPK is required for its inhibition.. Possik et al., (2014) also found that deletion of flcn-1 in nematode worms leads to ...
APP is phosphorylated at multiple sites in the 47 amino acid C-terminal cytoplasmic domain (Suzuki et al., 1994). Phosphorylation of Thr668 is particularly important, because it induces conformational changes that affect APP function and metabolism (Ando et al., 2001; Ramelot and Nicholson, 2001). To understand the mechanism of APP phosphorylation at Thr668 in neuronal cells, in this study, we investigated the role of the kinases Cdk5 and JNK and of their scaffolding and activating proteins. Using dominant-negative strategies and small molecule inhibitors, we found that JNK, not Cdk5, phosphorylates APP in differentiating neuronal cells. By preventing the interaction of JIP-1 with JNK or APP, we established that this JNK scaffolding, APP-binding protein does not participate in APP phosphorylation in neurons under normal physiological conditions. Importantly, we found that JIP-3, another JNK adaptor protein, which does not directly interact with APP, participates in the generation of a large ...
TY - JOUR. T1 - Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. AU - Zaidi, Sayyed K.. AU - Sullivan, Andrew J.. AU - Medina, Ricardo. AU - Ito, Yoshiaki. AU - van Wijnen, Andre J. AU - Stein, Janet L.. AU - Lian, Jane B.. AU - Stein, Gary S.. PY - 2004/2/25. Y1 - 2004/2/25. N2 - Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. Here we show that the endogenous Yes-associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast-related transcription factor, and suppresses Runx2 transcriptional activity in a dose-dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2-YAP complexes. Consequently, recruitment of the YAP co-repressor to ...
p42/microtubule-associated protein kinase (p42mapk) is activated by tyrosine and threonine phosphorylation, and its regulatory phosphorylation is likely to be important in signalling pathways involved in growth control, secretion, and differentiation. Here we show that treatment of quiescent 3T3 cells with diverse agonists results in the appearance of an activity capable of causing the in vitro phosphorylation of p42mapk on the regulatory tyrosine and to a lesser extent on the regulatory threonine, resulting in enzymatic activation of the p42mapk. This p42mapk-activating activity is capable of phosphorylating a kinase-defective p42mapk mutant, thus confirming its activity as a kinase. ...
The splicing factor Sf3b is an integral part of U2 snRNP and plays an essential role during spliceosome assembly and recognition of the introns branch point. One of the components of SF3b, SF3b1, is known to be reversibly phosphorylated during splicing catalysis [3], suggesting that protein kinases play a role in the regulation of splicing. Previous studies have shown that cyclin E/CDK2 complexes associate with spliceosomal proteins in vivo, and that CDK2 phosphorylates SF3b1 in vitro [12, 22]. Here we provide evidence that the protein kinase DYRK1A phosphorylates SF3b1 in vitro and in vivo.. The N-terminal part of Sf3b1 harbours a large number of Thr/Pro dipeptide motifs within a 240-amino acid region preceding the carboxyterminal repeat domain (Fig. 1). Both DYRK1A and CDK2 are proline-directed kinases, i.e. they phosphorylate serine or threonine residues followed by a proline residue [15, 23]. It has been shown that cyclin E/CDK2 phosphorylates SF3b1 in vitro at multiple sites within the ...
1538 Because of its versatility (all types of substrates), robustness (Z,0.8) , and rapid performance (10 minutes), and its ease of use, the luminescence based Kinase GloTM, Kinase Glo PlusTM, and now Kinase Glo Max assay platform have gained wide acceptance in many drug screening programs for protein kinase inhibitors. It is applicable to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, protein, polymer, lipids, and sugars). It also detects additional phosphorylation sites of already existing phosphopeptide substrates by enzymes such as GSK-3 and CK1, and monitors the activity of kinases phosphorylating their substrates on multiple sites. Since the linear range of ATP is extended to 500 µM, it is feasible to screen libraries for compounds that are not only competitive with ATP but also for those that are non competitive which broaden the selection of inhibitors of both serine/threonine protein kinases as well as tyrosine protein kinases. The ...
The intracellular localization of the S. cerevisiae transcription factor SWI5 is cell cycle dependent. The protein is nuclear in G1 cells but cytoplasmic in S, G2, and M phase cells. We have identified SWI5s nuclear localization signal (NLS) and show that it can confer cell cycle-dependent nuclear entry to a heterologous protein. Located within or close to the NLS are three serine residues, mutation of which results in constitutive nuclear entry. These residues are phosphorylated in a cell cycle-dependent manner in vivo, being phosphorylated when SWI5 is in the cytoplasm and dephosphorylated when it is in the nucleus. As all three serines are phosphorylated by purified CDC28-dependent H1 kinase activity in vitro, we propose a model in which the CDC28 kinase acts directly to control nuclear entry of SWI5.
Tuberin exists as a phosphoprotein, the target of both serine/threonine and tyrosine kinases (31) . Our data are consistent with that of others, which indicate that at least one of these kinases is Akt, a downstream effector of the PI3K signaling pathway (33, 34, 35, 36) . Tuberin contains three recognition sites for Akt that are also potential 14-3-3 binding sites. Using a novel protein domain array, a tuberin peptide containing Ser939 bound to 14-3-3 in a phosphorylation-specific manner. All seven 14-3-3 isoforms recognized tuberin in GST-pull-down assays, and at least one of these, 14-3-3γ, recognized tuberin in fibroblasts (NIH3T3) as well as mammary (MCF-7) and kidney epithelial cells (TRKE) from mouse, human, and rat, respectively. The interaction between 14-3-3γ and tuberin was effectively competed with phosphorylated but not unphosphorylated Ser939 peptide. Endogenous tuberin could also be coimmunoprecipitated with 14-3-3 in kidney epithelial cell lysates. While in submission, our data ...
TY - CONF. T1 - Evidence for phospho-tyrosine independent signalling activity of STAT1. AU - Majoros, Andrea. AU - Cheon, HyenJoo. AU - Shukla, Priyank. AU - Vogl, Claus. AU - Schreiber, Robert. AU - Stark, George R. AU - Mueller, Mathias. AU - Decker, Thomas. N1 - Conference code: 28. PY - 2014. Y1 - 2014. N2 - The Jak-Stat signaling pathway regulates cellular responses to cytokines. Stat1 plays a crucial role in host defense by mediating the effects of interferons (IFNs). In the canonical signaling pathway, IFNs activate STAT1 through phosphorylation at Y701. This leads to the formation of dimers that are competent to translocate to the cell nucleus, bind DNA and stimulate expression of interferon induced genes (ISGs). As reported, Stat1 also participates in a non-canonical signaling pathway that is independent of Y701 phosphorylation. In the presented study, this possibility was analysed by investigation of antibacterial immunity in mice expressing mutant Stat1Y701F. Our work suggests a ...
Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.
An hepatocyte cell-adhesion molecule (cell-CAM105) was recently shown to be identical with the liver plasma-membrane ecto-ATPase. This protein has structural features of the immunoglobulin superfamily and is homologous with carcinoembryonic antigen proteins. We have cloned a cDNA encoding a new form of the cell-CAM105 which is a variant of the previously isolated clone. In addition to having a shorter cytoplasmic domain, the new isoform also has substitutions clustered in the first 130 amino acids of the extracellular domain. Both of these isoforms are expressed on the surface of hepatocytes with the shorter variant being the predominant form. The previously isolated cell-CAM105 (long form) has more potential phosphorylation sites than does the new isoform (short form). Both isoforms are found to be phosphorylated after incubation with [32P]phosphate in vitro, with the long form being phosphorylated to a significantly higher extent. This observed differential phosphorylation could be one of the ...
Immunoblot analysis. Synaptosomal samples were rapidly solubilized in 1-2% SDS (95°C), sonicated, and protein concentration was measured using BCA assay (Pierce, Rockford, IL), with bovine serum albumin as standard. Equal amounts of protein were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. Immunoblots were done with 1:500 dilutions of the following phosphorylation state-specific antibodies: P-site 1 antibody (G-257), P-site 3 antibody (RU19), P-site 4/5 antibody (G-526), and P-site 6 antibody (G-555). The specificity of these antibodies for their respective sites has been characterized previously (Czernik et al., 1991; Jovanovic et al., 1996). Total synapsin I was detected by immunoblotting with synapsin I-specific antibody (G-486; 1:500 dilution). Primary incubations were followed by incubation with125I-labeled anti-rabbit IgG (1:500 dilution; Amersham Pharmacia Biotech, Little Chalfont, UK). Blots were exposed to a PhosphorImager screen, and quantification of ...
PINK1‐mediated phosphorylation of Ub at Ser65 has dramatic consequences for Ub structure, and key processes in the Ub system, namely Ub attachment and removal.. It could be expected that phosphorylation of Ub would change its surface properties due to the addition of a negative charge. The obtained high‐resolution crystal structure and solution studies agree that the majority of phosphoUb is structurally similar to wt Ub. To our amazement, NMR studies showed a second, minor conformation of phosphoUb, which is in slow exchange with the major conformation. Strikingly, the minor conformation shows distinct hydrogen bonding patterns and long‐range NOEs for its C‐terminal β5‐strand, which can only be structurally satisfied when this strand is shifted by two residues. Our phosphoUbretraCT model explains numerous observations and is structurally feasible due to the existence of four Leu‐Xaa repeats in the β5‐strand that would allow a shift of two residues without significantly ...
Phosphorylation is an important covalent post-translational modification (PTM) in cell signalling pathways. Protein phosphorylation is the reversible addition of a phosphate group to a protein or small molecule catalysed by protein kinases. Approximately one third of the 30,000 proteins encoded by the human genome contain covalently bound phosphate. The average protein kinase can add phosphates to 20 different proteins and the average protein phosphatase removes phosphate from 60 different proteins.
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Cytoplasmic expression of claudin-1 in metastatic melanoma cells correlates to increased migration, and increased secretion of MMP-2 in a PKC dependent manner, whereas claudin-1 nuclear expressi...
We statement here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. phosphorylation signals from protein manifestation changes thus providing a powerful tool to accurately profile cellular transmission transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and finding new signaling occasions. Protein phosphorylation one of the most ubiquitous post-translational adjustments continues to be implicated in the legislation of virtually all areas of a cells lifestyle. Aberrant phosphorylation dynamics inside the cell donate to the advancement and onset of several malignances.1 Therefore considerable work has been specialized in profiling proteins phosphorylation under Tm6sf1 different cellular circumstances. Currently most studies survey phosphorylation occasions that neglect to differentiate adjustments in phosphorylation from ...
Ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) is regulated at the transcriptional level during the cell cycle and in developing zebrafish embryos. We identify phosphorylation as a novel means of regulating UHRF1 and demonstrate that Uhrf1 phosphorylation is required for gastrulation in zebrafish. Human UHRF1 contains a conserved cyclin dependent kinase 2 (CDK2) phosphorylation site at serine 661 that is phosphorylated in vitro by CDK2 partnered with Cyclin A2 (CCNA2), but not Cyclin E. A phosphoserine-661 specific antibody recognizes UHRF1 in both in mammalian cancer cells and in non-transformed zebrafish cells, but not in zebrafish bearing a mutation in ccna2. Depleting Uhrf1 from zebrafish embryos by morpholino injection causes arrest before gastrulation and early embryonic death. This phenotype is rescued by wild-type UHRF1, but not by UHRF1 in which the phospho-acceptor site is mutated, demonstrating that UHRF1 phosphorylation is essential for embryogenesis. UHRF1 was ...
TY - JOUR. T1 - Pro-tumorigenic phosphorylation of p120 catenin in renal and breast cancer. AU - Kourtidis, Antonis. AU - Yanagisawa, Masahiro. AU - Huveldt, Deborah. AU - Copland, John A.. AU - Anastasiadis, Panos Z.. PY - 2015/6/11. Y1 - 2015/6/11. N2 - Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120), which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC) in the context of tumor progression, little is known about the status and ...
Phospholamban (PLN) regulates myocyte calcium cycling by inhibiting the Ca2+ATPase SERCA2a. Protein kinase A (PKA) mediated phosphorylation attenuates PLN activity leading to enhanced calcium uptake rates and accelerated cardiac relaxation. In vivo, PLN is present in monomeric and pentameric form. It is believed that PKA primarily targets the PLN monomer. However, we found that a R9C mutant of PLN dominantly inhibits PLN phosphorylation only within the pentamer suggesting a significant role of the pentamer in determining phosphorylation and, thus, PLN activity.. To investigate the role of the pentamer in PLN phosphorylation and function, the sensitivity, kinetics and stoichiometry of phosphorylation were analyzed in monomeric and pentameric PLN mutants expressed in a human cell line (HEK293AD). We found an independent increase of phosphorylation for monomer and pentamer upon forskolin stimulation, both in a concentration and time-dependent manner. Intriguingly, phosphorylation signals of PLN ...
Procaspase-8, the zymogen type of the apoptosis-initiator caspase-8, undergoes phosphorylation following integrin-mediated cell connection to an extracellular matrix base. CrkII and Crk, each bearing an Src-homology 2 domains (SH2) and one or two Src homology 3 (SH3) websites, respectively. CrkL (and knockouts display cardiac and sensory crest flaws, ending in embryonic lethality.17,18 Here, we offer proof that caspase-8 interacts with the You will need2 domains of CrkL in a Src- and adhesion-dependent way, and that this connections stimulates cellular migration. Outcomes Caspase-8 interacts with CrkL SH2 domains We observed the de novo phosphorylation of many protein, in caspase-8 showing cells selectively, pursuing cell adhesion to fibronectin substrates. These included a phosphoprotein at ~37 kDa (Fig.?1A). To determine whether the phosphoprotein may end up being component of a complicated linked with the caspase, caspase-8 immunoprecipitations had been performed by us, solved the necessary ...
The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cχ (PLCχ), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22- derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2·Sos complex was ...
The mutant c-erbB-2 protein with Glu instead of Val-659 exhibited transforming activity in NIH 3T3 cells. This protein showed enhanced tyrosine kinase activity in vitro and enhanced autophosphorylation at Tyr-1248 located proximal to the carboxyl terminus. Enhanced tyrosine phosphorylation of several cellular proteins was detected in cells expressing the Glu-659 c-erbB-2 protein. Introduction of an additional mutation at the ATP-binding site (Lys-753 to Met) of this protein resulted in abolition of its transforming ability. These data indicate that the transforming potential of c-erbB-2 is closely correlated with elevated tyrosine kinase activity of the gene product. To investigate the role of autophosphorylation in cell transformation, we introduced an additional mutation at the autophosphorylation site of the Glu-659 c-erbB-2 protein (Tyr-1248 to Phe). This mutant protein exhibited lower tyrosine kinase activity and lower transforming activity. On the other hand, when the carboxyl-terminal 230 ...
The invasion-promoting effect of ET-18-OMe on MCF-7/AZ cells suggests that ET-18-OMe initiates cSrc-mediated signalling in MCF-7/AZ cells, but not in the variant MCF-7/6 cells (Figure 1). Therefore the effect of ET-18-OMe on phosphorylation of Tyr397 of FAK, the autophosphorylation site of FAK, and Tyr416 of cSrc kinase was examined. Expression levels of FAK and cSrc in both cell lines were unchanged, but kinase activity of cSrc (Figures 2A, left-hand panel and 2B) and FAK (Figures 2A, left-hand panel and 2C) were greatly enhanced in MCF-7/AZ cells 5-10 min after treatment. There was no such activation of cSrc and FAK in MCF-7/6 cells (Figure 2A, right-hand panel). The use of cSrc kinase inhibitor, PP1, blocked activation of cSrc but not Tyr397 phosphorylation on FAK, suggesting that the autophosphorylation of FAK promotes activation of cSrc. Next, the cSrc-dependent tyrosine phosphorylation sites on FAK (Tyr576, Tyr861 and Tyr925) were assayed. Time-dependent phosphorylation of FAK on Tyr925 ...
Catalytic domain of the Protein Serine/Threonine Kinase, MAP/ERK kinase kinase 3. Serine/threonine kinases (STKs), MAP/ERK kinase kinase 3 (MEKK3) subfamily, catalytic (c) domain. STKs catalyze the transfer of the gamma-phosphoryl group from ATP to serine/threonine residues on protein substrates. The MEKK3 subfamily is part of a larger superfamily that includes the catalytic domains of other protein STKs, protein tyrosine kinases, RIO kinases, aminoglycoside phosphotransferase, choline kinase, and phosphoinositide 3-kinase. MEKK3 is a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK or MKKK or MAP3K), that phosphorylates and activates the MAPK kinase MEK5 (or MKK5), which in turn phosphorylates and activates extracellular signal-regulated kinase 5 (ERK5). The ERK5 cascade plays roles in promoting cell proliferation, differentiation, neuronal survival, and neuroprotection. MEKK3 plays an essential role in embryonic angiogenesis and early heart development. In addition, MEKK3 is ...
A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen- activated protein kinase (MAPK) p38 signaling cascade. The stable β-globin mRNA was rendered unstable by insertion of the 2,500-nucleotide Cox-2 3 untranslated region (3 UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38. This stabilization was blocked by SB203580, an inhibitor of p38, and by two different dominant negative forms of MAPK-activated protein kinase 2 (MAPKAPK-2), a kinase lying downstream of p38. Constitutively active MAPKAPK- 2 was also able to stabilize chimeric β-globin-Cox-2 transcripts. The MAPKAPK-2 substrate hsp27 may be involved in stabilization, as β-globin-Cox- 2 transcripts were partially stabilized by phosphomimetic mutant forms of hsp27. A short (123-nucleotide) fragment of the Cox-2 3 UTR was necessary and sufficient for the regulation of mRNA stability by the p38
Protein phosphorylation affects most, if not all, cellular activities in eukaryotes and is essential for cell proliferation and development. An estimated 30% of cellular proteins are phosphorylated, representing the phosphoproteome, and phosphorylation can alter a proteins function, activity, local …
Many stimuli mediate activation and nuclear translocation of ERK (extracellular-signal-regulated kinase) by phosphorylation on the TEY (Thr-Glu-Tyr) motif. This is necessary to initiate transcriptional programmes controlling cellular responses, but the mechanisms that govern ERK nuclear targeting are unclear. Single-cell imaging approaches have done much to increase our understanding of input-output relationships in the ERK cascade, but few studies have addressed how the range of ERK phosphorylation responses observed in cell populations influences subcellular localization. Using automated microscopy to explore ERK regulation in single adherent cells, we find that nuclear localization responses increase in proportion to stimulus level, but not the level of TEY phosphorylation. This phosphorylation-unattributable nuclear localization response occurs in the presence of tyrosine phosphatase and protein synthesis inhibitors. It is also seen with a catalytically inactive ERK2-GFP (green fluorescent ...
We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr139. Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr139, and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P3 or PtdIns(3,4)P2, is not phosphorylated at Tyr139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr139 of ...
Interestingly, recent in vitro kinetic studies using recombinant active p38α expressed in Escherichia coli showed that p38 phosphorylates GST-ATF2 (amino acids 1-115) via a two‐step (double collision) mechanism, involving the dissociation of mono‐phosphorylated ATF2 Thr71 or Thr69 from the enzyme after the first phosphorylation step (Waas et al., 2001). Moreover, these authors found that mono‐phosphorylation of ATF2 Thr69 strongly reduces the phosphorylation rate of Thr71, whereas, in contrast, mono‐phosphorylation of Thr71 does not reduce the rate of Thr69 phosphorylation. Thus, efficient phosphorylation of ATF2 by recombinant E.coli‐expressed active p38 only occurs in the order Thr71→ Thr69 + 71 (Figure 7). This order of events also seems to occur in mitogen‐treated cells, as ERK, in contrast to p38, does not seem to mono‐phosphorylate Thr69 significantly (Figure 4C).. The fact that ERK does not double‐phosphorylate ATF2 Thr69 + 71 efficiently raises the question as to ...
Phosphorylation in the activation segment of protein kinases is a common mechanism of kinase regulation. However, activation loop phosphorylation of many kinases generally induces activating structural changes by repositioning key structural elements that permit substrate and cofactor binding and efficient catalysis (51). Although no common mechanism has been proposed for negative regulation of protein-Ser/Thr kinases, phosphorylation of several of the CDKs within the subdomain I GXGXXG motif at the Thr14 and Tyr15 (human CDK1 numbering) are known to be inhibitory (67-69), and acetylation of the ATP coordinating Lys has been shown to reduce the kinase activity of CDK9 (70).. Here, we establish for the first time that mimicking phosphorylation of PLK1 on Tyr217 in the P+1 loop completely inhibits detectable kinase activity, likely through inhibition of substrate binding, although we cannot formally rule out the possibility that the effect is due to the Glu substitution rather than a ...
Cyclin-afhængig kinase 1 (Cdk1) er aktiveret i G2 fase af cellecyklus og regulerer mange cellulære veje. Her præsenterer vi en protokol ...
Serine-proline or threonine-proline is minimally required for Cdk-dependent phosphorylation (Errico, 2010), and we see that Ascl1 undergoes cell cycle-dependent phosphorylation in Xenopus egg extracts on these sites (Fig. 1; supplementary material Fig. S2); a mutant in which SP sites have been mutated to alanine-proline shows a dramatic reduction in phosphorylation.. To determine whether Ascl1 can indeed act as a target for Cdks, we incubated Ascl1 protein with active recombinant cyclin/Cdk proteins. When wild-type Ascl1 was incubated with CyclinA/Cdk2, its migration on SDS-PAGE was significantly retarded, and a smear of slower-migrating Ascl1 protein indicates phosphorylation on more than one site (supplementary material Fig. S3). S-A Ascl1 in this assay shows markedly reduced retardation compared with wild-type Ascl1, demonstrating that phosphorylation of Ascl1 occurs on SP sites. It is interesting to note that in vitro when incubated with purified kinases, some phosphorylation of S-A Ascl1 ...
Title: Phosphorylation-Specific Prolyl Isomerase Pin1 as a new Diagnostic and Therapeutic Target for Cancer. VOLUME: 8 ISSUE: 3. Author(s):Greg Finn and Kun Ping Lu. Affiliation:Cancer Biology Program,Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 1030, Boston, MA 02115, USA.. Keywords:anaphase-promoting complex, Pin1 expression, cyclin D1, Hepatitis B virus, WW domain, Nuclear Magnetic resonance. Abstract: Proline directed phosphorylation is a key regulatory mechanism controlling the function of fundamental proteins involved in cell proliferation and oncogenic transformation. Recently, the identification of the phosphorylation dependent prolyl isomerase Pin1 has uncovered a distinct regulatory mechanism controlling protein function. Specifically, Pin1 controls the conversion of peptidyl proline bond conversion from cis to trans, only when the preceding serine or threonine is phosphorylated. The ...
TY - JOUR. T1 - Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation. AU - Ek-Vitorin, Jose F.. AU - King, Timothy J.. AU - Heyman, Nathanael S.. AU - Lampe, Paul D.. AU - Burt, Janis M.. PY - 2006/6. Y1 - 2006/6. N2 - Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control ...
Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) ...
Activation induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR). AID initiates the processes that carry out immunoglobulin diversity by deaminating cytosine residues within variable (V) and switch (S) regions on the Ig locus during active transcription. The resulting G:U mispairs can then be replicated or repaired by cellular repair mechanisms to give rise to isotype-switched and antigen-specific mature antibodies.; In this study I have identified two novel phosphorylation sites, serine 41 and serine 43, and demonstrated their importance in AID activity as well as confirmed the importance of serine 38 phosphorylation. Phosphorylation null mutants generated by replacing serine with alanine are much less active than wild-type AID, as is non-phosphorylated AID purified from E. coli. In contrast, phosphorylation charge mimic mutants generated by replacing serine with aspartic acid, are (3-4) fold more active than wild-type AID. ...
Activation induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR). AID initiates the processes that carry out immunoglobulin diversity by deaminating cytosine residues within variable (V) and switch (S) regions on the Ig locus during active transcription. The resulting G:U mispairs can then be replicated or repaired by cellular repair mechanisms to give rise to isotype-switched and antigen-specific mature antibodies.; In this study I have identified two novel phosphorylation sites, serine 41 and serine 43, and demonstrated their importance in AID activity as well as confirmed the importance of serine 38 phosphorylation. Phosphorylation null mutants generated by replacing serine with alanine are much less active than wild-type AID, as is non-phosphorylated AID purified from E. coli. In contrast, phosphorylation charge mimic mutants generated by replacing serine with aspartic acid, are (3-4) fold more active than wild-type AID. ...
TY - JOUR. T1 - CFTR activation. T2 - Additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. AU - Wilkinson, Daniel J.. AU - Strong, Theresa V.. AU - Mansoura, Monique K.. AU - Wood, Deborah L.. AU - Smith, Stephen S.. AU - Collins, Francis S.. AU - Dawson, David C.. PY - 1997/7. Y1 - 1997/7. N2 - To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, - 712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased ...
Infection by Dengue virus is a global health problem for which there have been challenges to obtaining a cure. Current vaccines can only be narrowly applied in ongoing clinical trials. We employed computational methods to predict therapeutic efficacy based on structure-function relationships between human host kinases and viral Nonstructural Protein 3(NS3) in an effort to understand the therapeutic effect of inhibitors of viral replication. Phosphorylation at each of two most evolutionarily conserved sites, S137 and T189 compared to the unphosphorylated state were studied with molecular dynamics and docking simulations. The simulations suggested that phosphorylation at S137 caused a greater structural change than phosphorylation at T189. Docking studies supported the idea that phosphorylation at S137 increased the binding affinity between NS3 and NS5,whereas, phosphorylation at T189 decreased it. The interaction of NS3 and NS5is essential for viral replication. Docking studies with the antiviral ...
PFOS induces Sertoli cell injury using testicular cells isolated from rodent testes, but it remains unknown if PFOS has similar effects in humans. Herein, we maintained human Sertoli cells in a mitotically active state in vitro, thus enabling transfection experiments that altered gene expression to explore the molecular mechanism(s) underlying toxicant-induced cell injury. Human Sertoli cells obtained from men at ages 15, 23, 36 and 40 were cultured in vitro. These differentiated Sertoli cells remained mitotically active when cultured in the presence of 10% FBS (fetal bovine serum), with a replication time of ~1-3 weeks. At ~80% confluency, they were used for studies including toxicant exposure, immunoblotting, immunofluorescence analysis, tight junction (TJ)-permeability assessment, and overexpression of BTB (blood-testis barrier) regulatory genes such as FAK and its phosphomimetic mutants. PFOS was found to induce Sertoli cell injury through disruptive effects on actin microfilaments and ...
TY - JOUR. T1 - c-Src enhances the spreading of src-/-fibroblasts on fibronectin by a kinase-independent mechanism. AU - Kaplan, Kenneth B.. AU - Swedlow, Jason R.. AU - Morgan, David O.. AU - Varmus, Harold E.. PY - 1995/6/15. Y1 - 1995/6/15. N2 - We have explored the role of the tyrosine kinase c-Src in cellular adhesion. Fibroblasts derived from src-/-mice (src-/-fibroblasts) exhibit a reduced rate of spreading on fibronectin. This defect is rescued by expression of wild-type chicken c-Src. Analyses of mutants suggest that c-Src increases the rate of cell spreading in src-/- fibroblasts through a kinase-independent mechanism requiring both the SH3 and SH2 domains. To further address the role of c-Src in adhesion, we examined the activity and subcellular distribution of c-Src during the adhesion of fibroblasts on fibronectin. We observed a transient increase in the specific kinase activity of c-Src accompanied by the partial dephosphorylation of the negative regulatory site Y527. Activation of ...
Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both invitro and invivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays invitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to ...
BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the
It has been observed that coincident with or immediately following IκBα degradation, p65 is phosphorylated at multiple residues, and these phosphorylation events are necessary for proper regulation of NF-κB function (45). The phosphorylation patterns of NF-κB proteins have not been characterized in T cell anergy, and so we asked whether aberrant phosphorylation was responsible for the defects in NF-κB function in anergic cells. An early step involves phosphorylation of p65 at Ser536 by the IKK complex (32-35), and it has been suggested that phosphorylation at this residue negatively regulates the kinetics of p65 nuclear translocation (33). We found that p65 is phosphorylated at Ser536 equivalently in both naive and anergic cells, which is consistent with our finding that p65 translocates to the nucleus with normal kinetics in anergic T cells. A second posttranslational modification important for NF-κB activity is phosphorylation at Ser276. We found that, as with Ser536 phosphorylation, p65 ...
PURPOSE: To study in both in situ and primary cultures the posttranslational phosphorylation of connexin46 (Cx46), one of two members of the connexin family of gap junction proteins expressed by lens fibers. METHODS: Phosphatase digestion, gel electrophoresis, cell culture, organ culture, immunoprecipitation, metabolic labeling, and phosphoamino acid analysis were the methods used in this study. RESULTS: Cx46 immunoprecipitated from either rat or bovine lenses resulted in a shift to a more rapidly migrating species. During rat embryonic development, the more rapidly migrating, nonphosphorylated form of Cx46 was prevalent at 15 days gestation; as development progressed, there was a loss of the nonphosphorylated form with a concomitant increase in the phosphorylated form, such that by 28 days after birth only the phosphorylated form was detectable. The rate of posttranslational phosphorylation was very slow compared to previously measured rates for connexin43. Primary cultures of rat embryonic ...
Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I(A) PI3K beta and p101-coupled class I(B) PI3K gamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110 beta and serine 1101 of p110 gamma). Like other class I(A) PI3K isoforms, autophosphorylation of p110 beta resulted in down-regulated PI3K beta lipid kinase activity. However, no inhibitory effect of p110 gamma autophosphorylation on PI3K gamma lipid kinase activity was observed. Moreover, PI3K beta and PI3K gamma differed in the regulation of their autophosphorylation. Whereas p110 beta autophosphorylation was stimulated neither by G beta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived ...
Greiser and colleagues (10) also report that, consistent with previous studies, the remaining RyR2 clusters were hyperphosphorylated at the protein kinase A (PKA) phosphorylation site (Ser2808), which may compensate for the reduction in RyR2 protein expression and help sustain subsarcolemmal Ca2+ release despite reduced L-type Ca2+ currents (Figure 1B). However, RAP myocytes exhibited reduced RyR2 phosphorylation at the calmodulin-dependent protein kinase II (CaMKII) phosphorylation site (Ser2815) and no changes in CaMKII activity. This finding contrasts with previous studies that reported increased atrial CaMKII activity and CaMKII-dependent RyR2-Ser2815 phosphorylation in human AF (5). Moreover, other studies have shown that treatment with CaMKII inhibitors or selective disruption of the Ser2815 CaMKII phosphorylation site prevented AF in animal models through a reduction of SR Ca2+ leak (12). One explanation for this discrepancy could be the limited duration of pacing in the rabbit model used ...
Sun QY.,Wu GM.,Lai LX.,Bonk A.,Cabot R.,...&Schatten H.(2002).Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro.Biology of Reproduction,66(3),580-588 ...
Stimulation of hemopoietic cells with IL-3, IL-4, IL-5, granulocyte-macrophage-CSF and Steel factor-(SLF) induced tyrosine phosphorylation of a number of protein substrates. Two of these proteins, designated p42 and p44, were tyrosine phosphorylated rapidly in response to treatment with IL-3, IL-5, granulocyte-macrophage-CSF and SLF, but not IL-4. We demonstrate that these common substrates are members of the mitogen-activated protein kinase (MAP kinase) family of protein serine/threonine kinases. Ion-exchange chromatography yielded a peak of MAP kinase activity eluting at 0.3 to 0.32 M NaCl. Immunoblotting of column fractions with antiphosphotyrosine antibodies showed coelution of the peak of MAP kinase enzyme activity with the p42 and p44 tyrosine phosphorylated species, and with two proteins of 42 and 44 kDa which were immunoreactive with anti-MAP kinase antibodies. Moreover, a characteristic shift in mobility of the p42 and p44 species was observed after factor treatment. Time-course ...
The results of the present study identify the ERK 1/2 MAP kinase as being responsible for phosphorylation of eNOS at Ser116 in endothelial cells under basal conditions. Ser116 phosphorylation has been shown previously by Kou et al to be reduced by the protein kinase C (PKC) inhibitor calphostin C, implicating PKC as a mediator of this specific phosphorylation reaction.21 However, Shaw and colleagues22,23 have recently shown that the AGC kinases (protein kinase A, protein kinase G, and PKC), as well as the calmodulin-dependent protein kinases, cannot phosphorylate serines or threonines in protein substrates containing a proline at the P+1 position. Proline at P+1 is thus a veto residue that precludes phosphorylation by AGC and calmodulin-dependent protein kinases. This feature of proline-directed phosphorylation provides very tight control in preventing reciprocal substrate specificity between proline-directed protein kinases and AGC/calmodulin-dependent protein kinases. Because Ser116 in the ...
The large extracellular domain of glycoprotein hormone receptors is a unique feature within the G protein-coupled receptors (GPCRs) family. After interaction with the hormone, the receptor becomes coupled to Gs, which, in turn stimulates adenylyl cyclase and the production of cAMP. Potential phospho …
TY - JOUR. T1 - Serum response factor MADS box serine-162 phosphorylation switches proliferation and myogenic gene programs. AU - Iyer, Dinakar. AU - Chang, David. AU - Marx, Joe. AU - Wei, Lei. AU - Olson, Eric M.. AU - Parmaceki, Michael S.. AU - Balasubramanyam, Ashok. AU - Schwartz, Robert J.. PY - 2006/3/21. Y1 - 2006/3/21. N2 - Phosphorylation of a cluster of amino acids in the serum response factor (SRF) MADS box αI coil DNA binding domain regulated the transcription of genes associated with proliferation or terminal muscle differentiation. Mimicking phosphorylation of serine-162, a target of protein kinase C-α, with an aspartic acid substitution (SRF-S162D) completely inhibited SRF-DNA binding and blocked α-act in gene transcription even in the presence of potent myogenic cofactors, while preserving c-fos promoter activity because of stabilization of the ternary complex via Elk-1. Introduction of SRF-S162D into SRF null ES cells permitted transcription of the c-fos gene but was ...
We examined the sites of phosphorylation and the role that phosphorylation plays in the function of the M2-1 protein in transcription and interaction with viral RNA. The M2-1 protein was found by proteolytic digestion and site-directed mutagenesis to be phosphorylated at serine 58 and serine 61. Serines 58 and 61 lie in the consensus sequence for phosphorylation by CKI and are conserved in human, bovine, and ovine RS virus and in turkey rhinotracheitis virus (1, 8, 26, 29, 35, 37). The conservation of these residues suggests a functional role for M2-1 phosphorylation. Phosphorylation of serine 58 creates a new CKI site at serine 61. The disruption of serine 58 should prevent phosphorylation at serine 61, which would result in the loss of both phosphorylation sites, which is consistent with our results. Based on sequence analysis, mutation at serine 61 would be expected to affect phosphorylation at only that site and not at serine 58, explaining why the S61A mutant retains 33P incorporation. The ...
CHOP, a member of the C/EBP family of transcription factors, mediates effects of cellular stress on growth and differentiation. It accumulates under conditions of stress and undergoes inducible phosphorylation on two adjacent serine residues (78 and 81). In vitro, CHOP is phosphorylated on these residues by p38 mitogen-activated protein kinase (MAP kinase). A specific inhibitor of p38 MAP kinase, SB203580, abolished the stress-inducible in vivo phosphorylation of CHOP. Phosphorylation of CHOP on these residues enhanced its ability to function as a transcriptional activator and was also required for the full inhibitory effect of CHOP on adipose cell differentiation. CHOP thus serves as a link between a specific stress-activated protein kinase, p38, and cellular growth and differentiation. ...
OBJECTIVE: The posttranslational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites.. METHODS AND RESULTS: Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167, and threonine (T) 231 in HEK293 cells, whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51, S72, T85, T91, T103, S130, S167 and T231). GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2. ...
© the author(s), publisher and licensee Libertas Academica Ltd. Here we show by western blotting that transcriptionally active isoforms of p63 (p63α and p63γ) induce the phosphorylation of human 2N4R tau at the tau-1/AT8 epitope in HEK293a cells; a phospho-epitope increased in Alzheimers disease. Confocal microscopy shows that tau and p63α are spatially separated intracellularly. Tau was found in the cytoskeletal compartment, whilst p63α was located in the nucleus, indicating that the effects of p63 on tau phosphorylation are indirectly mediated. Tau phosphorylation occurred independently of the known tau kinases, protein kinase C delta (PKCδ), c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), glycogen synthase kinase 3 (GSK3), v-akt murine thymoma viral oncogene homolog (AKT) and cyclin-dependent kinase 5 (Cdk5) and the tau protein phosphatases (PP), PP1 and PP2A-Aαa/β. Considering that p63 and tau are both associated with
Phosphorylation-dependent interaction between antigenic peptides and MHC class I Curator: Larry H. Bernstein, MD, FCAP Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self. Mohammed F1, Cobbold M, Zarling AL, Salim M, Barrett-Wilt GA, Shabanowitz J, Hunt DF, Engelhard VH, Willcox BE. Nat Immunol.…
Aberrant activation of the intracellular PI3K-AKT-mTOR signaling pathway, which regulates critical processes such as cell cycle and survival, is one of the most common occurrences in human cancers and has been the focus of targeted therapy development. However, inhibitors targeting PI3K, AKT, or mTOR have shown limited clinical benefit. To identify new regulators of the PI3K-AKT pathway, Wheeler and colleagues screened 7,450 shRNAs for kinases or GTPases that affect AKT phosphorylation at serine 473 (S473), and identified 29 genes that had not been previously implicated in PI3K-AKT signaling, as well as genes known to regulate AKT phosphorylation. Of the 29 genes, the most-represented functional group was the RAB GTPases, which regulate endomembrane trafficking. Knockdown of RAB35, one of the five RAB GTPases identified in the screen, in multiple cell lines resulted in decreased AKT phosphorylation at S473 as well as diminished phosphorylation of phosphoinositide-dependent kinase 1 (PDK1) and ...
Protein kinases are able to recognize their appropriate targets in a complex milieu of cellular protein. This process must be carried out with high fidelity to ensure proper signal transduction in eukaryotic cells (Hunter 2000). In this study, we attempted to obtain insight into this recognition by examining PKA variants that exhibit a stable association with substrates. This binding provided a facile assay that allowed us to identify domains in both enzyme and substrates that were important for PKA phosphorylation. The substrate domains identified were physically removed from the sites of phosphorylation and were required for efficient recognition by PKA both in vivo and in vitro. To the best of our knowledge, these studies are the first to show that such distal sequence elements in substrates are required for phosphorylation by PKA. These observations may help explain why only a fraction of proteins that contain a PKA consensus site are phosphorylated by this enzyme in vivo (Budovskayaet al. ...
Quantitative phosphoproteomics workflows traditionally involve additional sample labeling and fractionation steps for accurate and in-depth analysis. Here we report a high-throughput, straightforward, and comprehensive label-free phosphoproteomics approach using the highly selective, reproducible, and sensitive Ti(4+)-IMAC phosphopeptide enrichment method. We demonstrate the applicability of this approach by monitoring the phosphoproteome dynamics ... read more of Jurkat T cells stimulated by prostaglandin E2 (PGE2) over six different time points, measuring in total 108 snapshots of the phosphoproteome. In total, we quantitatively monitored 12,799 unique phosphosites over all time points with very high quantitative reproducibility (average r , 0.9 over 100 measurements and a median cv , 0.2). PGE2 is known to increase cellular cAMP levels, thereby activating PKA. The in-depth analysis revealed temporal regulation of a wide variety of phosphosites associated not only with PKA, but also with a ...
We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAKTyr407, but not ...
The mechanism of activation for protein kinase B (PKB), an important target for insulin signaling, has been scarcely investigated in primary cells. In this study, we have characterized the insulin-induced phosphorylation and activation of PKB beta in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKB beta from cytosol to membranes, and phosphorylation and activation of PKB beta. Phosphoamino acid analysis and phosphopeptide mapping demonstrated that the phosphorylation occurred mainly on serines, also when using calyculin A, and that these were localized within one major phosphopeptide. Radiosequencing showed that the radioactivity was released in Cycle No. 7. In addition, the peptide was specifically immunoprecipitated from a tryptic digest of PKB beta using the anti-phospho-PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKB beta mainly is phosphorylated on Ser-474 in response to insulin stimulation, in contrast to previous studies in ...
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The purpose of this study was to determine whether phosphorylation has an effect on the characteristics of the 60 kD Ro antigen throughout the cell cycle. Cell extracts of synchronized HEp-2 cells were phosphorylated in vitro with exogenous ATP, exam
Supplementary MaterialsSupplementary Document. phosphorylated at tyrosine 97 in the postischemic mind upon neuroprotective insulin treatment, but how such posttranslational changes affects mitochondrial rate of metabolism is unclear. Here, we report the structural features and functional behavior of a phosphomimetic cytochrome mutant, which was generated by site-specific incorporation at position 97 of oxidase, or complex IV, within respiratory supercomplexes was higher than that of the wild-type species, in agreement with the observed decrease in reactive oxygen species production. Direct contact of cytochrome with the respiratory supercomplex factor HIGD1A (hypoxia-inducible domain family member 1A) is reported here, with the mutant heme protein exhibiting a lower affinity than the wild-type species. Interestingly, phosphomimetic cytochrome also exhibited a lower caspase-3 activation activity. Altogether, these findings yield a better understanding of the molecular basis for mitochondrial ...
Agonist-induced phosphorylation has been demonstrated for a variety of GPCRs including the β-adrenergic (Ferguson et al., 1995; Freedman et al., 1995; Fredericks et al., 1996; January et al., 1997), α-adrenergic (Easonet al., 1995), δ-opioid (Pei et al., 1995), endothelin (Freedman et al., 1997), adenosine (Palmeret al., 1995), vasopressin (Innamorati et al., 1997), and somatostatin (Hipkin et al., 1997) receptors. However, there have been relatively few unequivocal reports of AT1-R phosphorylation. This has been due in large part to the inability to distinguish the immunoprecipitated phospho-AT1-R from more abundant phosphoproteins that either genuinely or spuriously coprecipitate with the receptor (Smith et al., 1998). Despite these problems, unequivocal agonist-induced phosphorylation of a transiently expressed epitope-tagged AT1-R (Oppermann et al., 1996), and of a stably expressed (His)6-tagged AT1-R (Balmforth et al., 1997) has been reported in human embryonic kidney 293 cells. We ...
Functional selectivity, which highlights the ability of ligands to differentially activate the signalling pathways linked to G protein-couple receptors (GPCRs) has provided an avenue for developing ligands with greater safety profiles. Pilocarpine (Pilo), a non-selective muscarinic acetylcholine receptor (mAChR) agonist has been shown to differentially activate G protein subtypes linked to the M3 mAChR. In this study the pharmacology of Pilo was further investigated using a number of readouts. When compared to methacholine (MCh), a reference agonist, Pilo appeared to preferentially stimulate inositol phosphates production than global receptor phosphorylation. The ligand also appeared to preferentially promote phosphorylation of Ser412 at the third intracellular loop of the receptor than Ser577 at the C-terminal tail. This differential phosphorylation may be linked to the fact that these residues are phosphorylated by distinct protein kinases. However, such preferential phosphorylation was not ...
In this study, we investigated the requirements for NS5A phosphorylation. Taking advantage of the different phosphorylation patterns of NS5A observed with two cloned full-length genomes, a genetic analysis was performed. Our results show that a continuous NS3-5A sequence is required for NS5A hyperphosphorylation. Mutations at various positions in the NS3-4B region, not affecting polyprotein processing, can reduce or enhance this NS5A modification. Thus, structural integrity of each of these proteins, forming most likely a multisubunit protein complex, is essential for differential phosphorylation of NS5A.. Although phosphorylation of NS5A is a biochemical trait conserved among all HCV isolates analyzed so far, the conditions required for this modification appear to differ between various genotypes and even between different isolates of the same genotype. In case of the genotype 1a HCV-H isolate, the phosphorylation patterns of NS5A expressed on its own or in the context of an NS2-5B polyprotein ...
G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast. A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo. A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication. Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction. These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast. ...
The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained
BioAssay record AID 1299281 submitted by ChEMBL: Induction of p53 phosphorylation at serine-15 in human p53 +/+ HCT 116 cells bat 5 or 10 uM after 24 hrs by western blot analysis.
Protein phosphorylation often activates (or deactivates) many enzymes. Phosphorylation of sugars is often the first stage in ... While phosphorylation is performed by ATPs during preparatory steps, phosphorylation during payoff phase is maintained by ... Protein phosphorylation is the most abundant post-translational modification in eukaryotes. Phosphorylation can occur on serine ... Thr and Tyr phosphorylation. The prominent role of protein phosphorylation in biochemistry is illustrated by the huge body of ...
Phosphorylation of the enzyme GSK-3 by AKT (Protein kinase B) as part of the insulin signaling pathway. Phosphorylation of Src ... Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine is thought to be the most common ... Tyrosine phosphorylation is relatively rare but lies at the head of many protein phosphorylation signalling pathways (e.g. in ... Histidine phosphorylation of eukaryotic proteins appears to be much more frequent than tyrosine phosphorylation. In prokaryotic ...
... (UK /ɒkˈsɪd.ə.tɪv/, US /ˈɑːk.sɪˌdeɪ.tɪv/ ) or electron transport-linked phosphorylation or terminal ... This phosphorylation reaction is an equilibrium, which can be shifted by altering the proton-motive force. In the absence of a ... Oxidative phosphorylation uses these molecules and O2 to produce ATP, which is used throughout the cell whenever energy is ... The term oxidative phosphorylation was coined by Volodymyr Belitser [uk] in 1939. For another twenty years, the mechanism by ...
Regulation with tyrosine phosphorylation plays a very important role in gene regulation. Tyrosine phosphorylation can influence ... suggest that tyrosine phosphorylation may regulate synaptic transmission. The role of tyrosine phosphorylation in the ... Tyrosine phosphorylation is the addition of a phosphate (PO43−) group to the amino acid tyrosine on a protein. It is one of the ... leading to autophosphorylation and phosphorylation of several receptor substrates. Phosphorylation of selected tyrosine sites ...
A phosphorylation cascade is a sequence of signaling pathway events where one enzyme phosphorylates another, causing a chain ... MAP kinase not only plays an important function during growth of cell in the M phase phosphorylation cascade but also plays an ... The end product of a Phosphorylation cascade is the changes occurring inside the cell. One best example that explains this ... Denhardt, David T. (1996-09-15). "Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the ...
Unlike oxidative phosphorylation, oxidation and phosphorylation are not coupled in the process of substrate-level ... Most ATP is generated by oxidative phosphorylation in aerobic or anaerobic respiration while substrate-level phosphorylation ... producing 3-phosphoglycerate and ATP through a substrate-level phosphorylation. The second substrate-level phosphorylation ... Substrate-level phosphorylation is a metabolism reaction that results in the production of ATP or GTP by the transfer of a ...
Phosphorylation. It consists of the addition of a phosphate group (HPO3). In EHD3, there are two serine phosphorylations; one ... Apart from an EF-hand domain, it can also include tyrosine phosphorylation sites and coiled coils. This domain is often related ...
Furthermore, the second phosphorylation event is necessary to allow the formation of two charged groups (rather than only one) ... The phosphorylation and dephosphorylation of these enzymes (ultimately in response to the glucose level in the blood) is the ... A final substrate-level phosphorylation now forms a molecule of pyruvate and a molecule of ATP by means of the enzyme pyruvate ... This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and ...
Berg, Jeremy M.; Tymoczko, J. L.; Stryer, L. (2006). "Oxidative phosphorylation". Biochemistry (5 ed.). pp. 491-526. ISBN 978- ...
Whittam's research was on electrolyte and water in relation to metabolism; oxidative phosphorylation; the relationship between ...
Serine phosphorylation. Most of the seven STATs (except STAT2) undergo serine phosphorylation. Serine phosphorylation of STATs ... It has been proposed that phosphorylation of serine can regulate STAT1 dimerization, and that continuous serine phosphorylation ... Phosphorylation then recruits an adaptor protein called Shc, which activates the MAPK/ERK pathway, and this facilitates gene ... Liu, L.; McBride, K. M.; Reich, N. C. (2005). "STAT3 nuclear import is independent of tyrosine phosphorylation and mediated by ...
"Phosphorylation - US". www.thermofisher.com. Retrieved 2020-05-03. "ELM - Detail for TRG_NLS_Bipartite_1". elm.eu.org. ... Phosphorylation will affect different cellular processes and thus regulating protein function. SMIM15 has a transmembrane ... Finally, there are four predicted sites for phosphorylation of tyrosine on position 20, threonine on positions 25 and 31, and ...
For example, phosphorylation of TMEM128 may make it bind to different substrates through conformational change. TMEM128 also ... "Phosphorylation - US". www.thermofisher.com. Retrieved May 2, 2020. "Expression of TMEM128 in cancer - Summary - The Human ... phosphorylation, SUMOylation, and O-GlcNAc as seen below: Post-translational modification alters protein structure and can thus ...
Phosphorylation events, therefore, are controlled not only by the balanced activity of PKs and PPs but also by their restricted ... Phosphorylation involves the transfer of phosphate groups from ATP to the enzyme, the energy for which comes from hydrolysing ... Protein phosphorylation is one of the most common forms of reversible protein posttranslational modification (PTM), with up to ... Protein phosphorylation plays a crucial role in biological functions and controls nearly every cellular process, including ...
The biosynthesis of ATP is achieved throughout processes such as substrate-level phosphorylation, oxidative phosphorylation, ... "Oxidative phosphorylation". W H Freeman, 2002. Retrieved 4 April 2013. Medh, J. D. "Electron Transport Chain (Overview)" (PDF ... It is this energy coupling and phosphorylation of ADP to ATP that gives the electron transport chain the name oxidative ... Oxidative phosphorylation produces 26 of the 30 equivalents of ATP generated in cellular respiration by transferring electrons ...
A total of 13 likely phosphorylation sites were predicted: Ser: 5 Thr: 6 Tyr: 2 The main concentration of the phosphorylation ... ". "ExPASy Phosphorylation". "ExPASy Phyre2". "ExPASy SOSUI". Archived from the original on 2004-03-20. (Orphaned articles from ...
Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF (January 2002). "Phosphorylation of Ser307 in insulin receptor ... This protein then begins a phosphorylation cascade, culminating in the activation of mitogen-activated protein kinase (MAPK), ... Pearson RB, Kemp BE (1991). "Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations". ... Benaim G, Villalobo A (August 2002). "Phosphorylation of calmodulin. Functional implications". European Journal of Biochemistry ...
July 2002). "Formation of an hER alpha-COUP-TFI complex enhances hER alpha AF-1 through Ser118 phosphorylation by MAPK". The ... Lannigan DA (January 2003). "Estrogen receptor phosphorylation". Steroids. 68 (1): 1-9. doi:10.1016/S0039-128X(02)00110-1. PMID ... "Activation of estrogen receptor alpha by S118 phosphorylation involves a ligand-dependent interaction with TFIIH and ...
Utilizing one full oxygen in oxidative phosphorylation requires the transfer of four electrons. The oxygen will then consume ... "Oxidative phosphorylation revisited". Biotechnology and Bioengineering. 112 (3): 429-437. doi:10.1002/bit.25492. ISSN 1097-0290 ... 464 An electrochemical gradient is essential to mitochondrial oxidative phosphorylation. The final step of cellular respiration ... bacteriorhodopsin and noncyclic photophosphorylation and oxidative phosphorylation. The way bacteriorhodopsin generates a ...
Hashimoto Y, Katayama H, Kiyokawa E, Ota S, Kurata T, Gotoh N, Otsuka N, Shibata M, Matsuda M (July 1998). "Phosphorylation of ... Takahashi K, Suzuki K, Tsukatani Y (July 1997). "Induction of tyrosine phosphorylation and association of beta-catenin with EGF ... Olive DM (October 2004). "Quantitative methods for the analysis of protein phosphorylation in drug development". Expert Review ... There are several quantitative methods available that use protein phosphorylation detection to identify EGFR family inhibitors ...
Studies on phosphorylation. Part II. The reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. A new ... method for the phosphorylation of amines", Journal of the Chemical Society (Resumed) (in German), pp. 660-663, doi:10.1039/ ...
The dissertation was entitled "The influence of thyroidhormones in vivo on oxidative phosphorylation and enzyme activities in ... 1800, 205-212 (2010). B. Kadenbach (ed.): Mitochondrial Oxidative Phosphorylation. Nuclear-Encodes Genes, Enzyme Regulation, ... Subunit Composition and Phosphorylation Sites of Cytochrome c Oxidase: A trailblazer's tale - Bernhard Kadenbach". ... New extension of the Mitchell Theory for oxidative phosphorylation in mitochondria of living organisms, Biochim. Biophys. Acta ...
Minakami R, Jinnai N, Sugiyama H (1997). "Phosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype ... Benaim G, Villalobo A (2002). "Phosphorylation of calmodulin. Functional implications" (PDF). Eur. J. Biochem. 269 (15): 3619- ... interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein ...
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR (May 2000). "PKA phosphorylation dissociates ... Benaim G, Villalobo A (Aug 2002). "Phosphorylation of calmodulin. Functional implications". European Journal of Biochemistry. ...
Three phosphorylation sites were identified with conservation between human and mouse genotypes at T112-p, S114-p, and T-379-p ... "FAM43A phosphorylation sites". Phosphosite. Retrieved 8 April 2018. "FAM43A". IMP Bioinformatics. Retrieved 8 April 2018. " ... involved in the phosphorylation of SR splicing factors and the regulation of splicing. SRSF protein kinase 2 promotes neural ... apoptosis by up-regulating cyclin-D1 expression through the suppression of p53/TP53 phosphorylation. Protein phosphatase 2A is ...
Benaim G, Villalobo A (August 2002). "Phosphorylation of calmodulin. Functional implications". European Journal of Biochemistry ... interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein ...
Benaim G, Villalobo A (2002). "Phosphorylation of calmodulin. Functional implications". Eur. J. Biochem. 269 (15): 3619-31. doi ...
First, phosphorylation occurs. This creates a reactive intermediate, which in the second stage undergoes concerted ... diphosphate decarboxylase probably evolved from a common ancestor since they have a similar fold and catalyze phosphorylation ...
CSK acts by suppressing the activity of the Src family of protein kinases by phosphorylation of Src family members at a ... Y184F - Abolishes phosphorylation. Y304F - Decreases activity by two-thirds and alters conformation. S364A - Strong decrease of ... Upon phosphorylation by other kinases, Src-family members engage in intramolecular interactions between the phosphotyrosine ... "Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure". The ...
Twarog R, Wolfe RS (August 1962). "Enzymatic phosphorylation of butyrate". The Journal of Biological Chemistry. 237: 2474-7. ...
Combined oxidative phosphorylation deficiency 1 is a severe condition that primarily impairs neurological and liver function. ... Combined oxidative phosphorylation deficiency 1 is caused by mutations in the GFM1 gene. This gene provides instructions for ... Combined oxidative phosphorylation deficiency 1 is likely a rare disorder, although its prevalence is unknown. At least 12 ... Combined oxidative phosphorylation deficiency 1 is a severe condition that primarily impairs neurological and liver function. ...
News-Medical.Net provides this medical information service in accordance with these terms and conditions. Please note that medical information found on this website is designed to support, not to replace the relationship between patient and physician/doctor and the medical advice they may provide. ...
Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. ... Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation. ... Large-scale phosphorylation analysis of mouse liver Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1488-93. doi: 10.1073/pnas. ... Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. ...
Home FAQs DNA End Modification: Phosphorylation FAQ: DNA End Modification: Phosphorylation. Links to this resource. ...
IP7 phosphorylation appears selective for eukaryotic organisms, with none seen in Escherichia coli extracts. Phosphorylation ... ATP-free phosphorylation. For the first time, scientists have described a way for cells to add phosphate groups to proteins ... "Whats also remarkable is we have evidence not just of phosphorylation of proteins, but of pyrophosphorylation, adding a ...
In the present study, we demonstrated that FAK is implicated in RCP-induced EGFR phosphorylation and ovarian cancer cell ... Ectopic expression of RCP induced FAK phosphorylation, which links β1 integrin with EGFR and participates in a positive ... 4: Rab11 and Rab25 are required for RCP-induced FAK phosphorylation.. a, b SKOV-3 cells were co-transfected with the indicated ... 5: Curcumin inhibits stabilization of β1 integrin protein and FAK phosphorylation.. a SKOV-3 cells were transfected the ...
... and that process is what we call Oxidative Phosphorylation. Oxidative, Oxidative Phosphorylation. Now the main player, when ... You wouldnt be able to use it to actually do useful work, and so the process of Oxidative Phosphorylation is all about doing ... How oxidation of co-enzymes like NADH can lead to the production of ATP through oxidative phosphorylation. ... which can then be oxidized as part of Oxidative Phosphorylation. Other times, well actually, thats going to be attached to an ...
Oxidative phosphorylation - Arabidopsis thaliana (thale cress) [ Pathway menu , Organism menu , Pathway entry , Download KGML ...
We found that Pin1 could affect the phosphorylation of p38 MAPK, have no obvious difference in extracellular signal-regulated ... We found that Pin1 could affect the phosphorylation of p38 MAPK, have no obvious difference in extracellular signal-regulated ... change the conformation of proteins through protein phosphorylation, thus regulate various cellular processes in the body. Pin1 ... change the conformation of proteins through protein phosphorylation, thus regulate various cellular processes in the body. Pin1 ...
... Reversible protein phosphorylation plays a key role in signaling ... Tyrosine phosphorylation of proteins at two different stages in zebrafish embryonic development was studied by immuno-affinity ... We detected differential phosphorylation of known regulators of gastrulation cell movements, including Gravin, PDLIM5 and small ... Therefore we have explored the possibilities to use zebrafish development as a model system to study in vivo phosphorylation ...
A novel mechanism of regulation of phosphatidylinositol 3 -kinase: Tyrosine phosphorylation of p85 residue 688. ...
"Oxidative Phosphorylation Coupling Factors" is a descriptor in the National Library of Medicines controlled vocabulary ... This graph shows the total number of publications written about "Oxidative Phosphorylation Coupling Factors" by people in ... Below are the most recent publications written about "Oxidative Phosphorylation Coupling Factors" by people in Profiles. ... Below are MeSH descriptors whose meaning is more general than "Oxidative Phosphorylation Coupling Factors". ...
Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun. PloS one ... Gentisic acid induced ERK1/2 phosphorylation in HaCaT cells. HaCaT cells were treated with various concentration of gentisic ... Gentisic Acid Stimulates Keratinocyte Proliferation through ERK1/2 Phosphorylation Minho Kim1, JaeGoo Kim1, Yu-Kyong Shin2, Ki- ... Gentisic Acid Stimulates Keratinocyte Proliferation through ERK1/2 Phosphorylation. Int J Med Sci 2020; 17(5):626-631. doi: ...
Gene expression values, collected using RNA sequencing, were analyzed for glycolysis, oxidative phosphorylation, and ... complex I and complex IV in oxidative phosphorylation, and within the secondary part of the Hippo Kinase Signaling cascade. ... Expression of Glycolysis, Oxidative Phosphorylation, and the Hippo Signaling Pathway in Preeclampsia. Undergraduate Honors ... Gene expression values, collected using RNA sequencing, were analyzed for glycolysis, oxidative phosphorylation, and ...
phosphorylation ; hotspots ; hotspot ; kinase ; PTMs ; Protein kinase ; Ubiquitination ... These regions of conserved phosphorylation, defined as phosphorylation hotspots, were determined us- ing phosphosite data for a ... Structural analysis of phosphorylation hotspots and kinase target preferences Author: Strumillo, Marta Julia ORCID: 0000-0003- ... To better predict phosphorylation of high functional relevance I analysed phosphosites that are highly conserved across species ...
Nevertheless, we demonstrate here temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC ... No Transcription-Translation Feedback in Circadian Rhythm of KaiC Phosphorylation *Tomita, Jun ... Nevertheless, we demonstrate here temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC ...
Crystal Structure of Full Length Circadian Clock Protein KaiC with Correct Geometry at Phosphorylation Sites ... Crystal Structure of Full Length Circadian Clock Protein KaiC with Correct Geometry at Phosphorylation Sites. *PDB DOI: 10.2210 ... Crystal Structure of Full Length Circadian Clock Protein KaiC with Correct Geometry at Phosphorylation Sites. Pattanayek, R., ...
Protein Phosphorylation involves the addition of phosphate groups to proteins, most commonly serine (S), threonine (T) or ...
Jia J., Zhang L., Zhang Q., Tong C., Wang B., Hou F., Amanai K., Jiang J., Phosphorylation by double-time/CKIε and CKIα targets ... Chen Y., Sasai N., Ma G., Yue T., Jia J., Briscoe J., Jiang J., Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is ... PKA-mediated phosphorylation of Smo further enhanced its association with PKAc to form stable kinase-substrate complexes that ... Jia H., Liu Y., Yan W., Jia J., PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development ...
PI4KIIα phosphorylation by GSK3 directs vesicular trafficking to lysosomes James W. Robinson; James W. Robinson ... Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution ... Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic ... PI4KIIα phosphorylation by GSK3 directs vesicular trafficking to lysosomes. Biochem J 15 November 2014; 464 (1): 145-156. doi: ...
Hardwick, L. J., Azzarelli, R., & Philpott, A. (2018). Cell cycle-dependent phosphorylation and regulation of cellular ... Here, we focus on a direct mechanistic link involving phosphorylation of differentiation-associated transcription factors by ... Phosphorylation, Cell Lineage, Embryonic Development, Homeostasis, Transcriptome ...
Erratum for Bouzioukh et al., Tyrosine Phosphorylation Sites in ephrinB2 Are Required for Hippocampal Long-Term Potentiation ... Erratum for Bouzioukh et al., Tyrosine Phosphorylation Sites in ephrinB2 Are Required for Hippocampal Long-Term Potentiation ... Erratum for Bouzioukh et al., Tyrosine Phosphorylation Sites in ephrinB2 Are Required for Hippocampal Long-Term Potentiation ... Erratum for Bouzioukh et al., Tyrosine Phosphorylation Sites in ephrinB2 Are Required for Hippocampal Long-Term Potentiation ...
... Ghent University. Faculty of Sciences, 2019. ... Vu, L. D. (2019). Phosphorylation mediates high temperature signalling in plants. Ghent University. Faculty of Sciences, Ghent ... L. D. Vu, "Phosphorylation mediates high temperature signalling in plants," Ghent University. Faculty of Sciences, Ghent, ... Phosphorylation mediates high temperature signalling in plants. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2019. ...
Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide from p16CDKN2/INK4A by R. Fåhraeus et al. ... G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment. *S. ... Inhibition of pRb phosphorylation and cell cycle progression by an antennapedia-p16(INK4A) fusion peptide in pancreatic cancer ... Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide from p16CDKN2/INK4A. @article{ ...
A curated compendium of phosphorylation motifs. 2007, Pubmed Amanchy, A curated compendium of phosphorylation motifs. 2007, ... phosphorylation site. Figure S3. Heat map of all phosphorylation site changes. Related to Figure 4. (A) Full heat map of all ... Inserts of each panel identify the detected sites of phosphorylation. Figure 6. Consensus phosphorylation sites. GproX was used ... Heat map of all phosphorylation site changes. Related to Figure 4. &RKHQ¶V Analysis of individual phosphorylation sites ...
... using fluorescence-labelled peptides as substrate at 70 uM after 90 mins by microfluidic peptide phosphorylation assay. ... Inhibition of human FGFR2 N549H mutant catalytic domain expressed in baculovirus assessed as substrate phosphorylation ...
The H2A.X Phosphorylation Assay Kit (Flow cytometry) is a cell based assay formatted for flow cytometric detection of levels of ... Phosphorylation of the histone variant H2A.X is a rapid and sensitive response to double strand DNA breaks. The H2A.X ... The H2A.X Phosphorylation Assay Kit (Flow cytometry) is a cell based assay formatted for flow cytometric detection of levels of ... The H2A.X Phosphorylation Assay Kit (Flow cytometry) is a cell based assay formatted for flow cytometric detection of levels of ...
... Show full item record ... Functional Analysis of Phosphorylation on Saccharomyces cerevisiae Syntaxin 1 Homologues Sso1p and Sso2p. ... Yuan , Q & Jäntti , J 2010 , Functional Analysis of Phosphorylation on Saccharomyces cerevisiae Syntaxin 1 Homologues Sso1p ...
Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation. ... Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation ... Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation ...
Specifically, we wanted to determine whether increased phosphorylation (via Actn4S160D/S160D mice) or absent phosphorylation ( ... Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis. Di Feng, Mukesh Kumar, Jan Muntel ... Phosphorylation of ACTN4 Leads to Podocyte Vulnerability and Proteinuric Glomerulosclerosis. Di Feng, Mukesh Kumar, Jan Muntel ... 66 Although the above-mentioned phosphorylation events were associated with cellular changes, we did not detect phosphorylation ...
  • We found that Pin1 could affect the phosphorylation of p38 MAPK, have no obvious difference in extracellular signal-regulated kinases (ERK) and Jun-amino-terminal kinase (JNK) signaling. (frontiersin.org)
  • Tyrosine phosphorylation of proteins at two different stages in zebrafish embryonic development was studied by immuno-affinity purification and multi-dimensional LC-MS. Tyrosine kinases including members of the Src family of kinases, Eph receptors and Focal adhesion kinase as well as adaptor proteins such as Paxillin, Crk and p130Cas were identified. (uu.nl)
  • Western blot analysis of proteins in the mitogen-activated protein (MAP) kinase signaling pathway showed that ERK1/2 phosphorylation was increased by gentisic acid treatment. (medsci.org)
  • Significance testing demonstrated change in expression for the first step of glycolysis, complex I and complex IV in oxidative phosphorylation, and within the secondary part of the Hippo Kinase Signaling cascade. (unl.edu)
  • Protein Phosphorylation involves the addition of phosphate groups to proteins, most commonly serine (S), threonine (T) or tyrosine (Y) residue by a kinase and is essential for pathway activation in cellular regulation, cell signaling and growth. (gbiosciences.com)
  • Hedgehog binds to the transmembrane receptor Patched to relieve inhibition of the seven-transmembrane protein Smoothened (Smo), which in turn activates the transcription factor Ci by inhibiting its phosphorylation by the kinase PKA. (science.org)
  • PKA-mediated phosphorylation of Smo further enhanced its association with PKAc to form stable kinase-substrate complexes that promoted the PKA-mediated transphosphorylation of Smo dimers. (science.org)
  • Fascaplysin will prove to be a useful tool in studying the consequence of Cdk4 inhibition, especially in cells containing inactivated p16, and caused G1 arrest and prevented pRb phosphorylation at sites implicated as being specific for Cdk 4 kinase. (semanticscholar.org)
  • Age-associated impairment of Akt phosphorylation in primary rat hepatocytes is remediated by alpha-lipoic acid through PI3 kinase, PTEN, and PP2A. (oregonstate.edu)
  • Genes on mtDNA provide instructions for proteins that are primarily involved in the process of converting the energy from food into a form cells can use ( oxidative phosphorylation ). (medlineplus.gov)
  • As a result, fewer mitochondrial proteins involved in oxidative phosphorylation are produced. (medlineplus.gov)
  • The process of oxidative phosphorylation involves five groups of proteins, or complexes. (medlineplus.gov)
  • Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. (nih.gov)
  • What's also remarkable is we have evidence not just of phosphorylation of proteins, but of pyrophosphorylation, adding a phosphate on top of a phosphate. (the-scientist.com)
  • Pin1 is the only known peptidyl-prolyl cis-trans isomerase (PPIase) that can specifically recognize and isomerize the phosphorylated Serine/Threonine-Proline (pSer/Thr-Pro) motif, change the conformation of proteins through protein phosphorylation, thus regulate various cellular processes in the body. (frontiersin.org)
  • Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution throughout the cell. (portlandpress.com)
  • We detected the expected phosphorylation of MAP kinases, translational regulatory proteins, and subunits of APC/C that validate the accuracy of our measurements. (xenbase.org)
  • We find that more than half the identified proteins possess multiple sites of phosphorylation that are often clustered, where kinases work together in a hierarchical manner to create stretches of phosphorylated residues, which may be a means to amplify signals or stabilize a particular protein conformation. (xenbase.org)
  • Conversely, other proteins have opposing sites of phosphorylation that seemingly reflect distinct changes in activity during this developmental timeline. (xenbase.org)
  • Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. (strath.ac.uk)
  • Role of phosphorylation and dephosphorylation of proteins in the regulation of the metabolic activity of chloroplasts]. (uchicago.edu)
  • Fibroblast growth factor or nerve growth factor (NGF) stimulation leads to tyrosine phosphorylation of the FGF Receptor Substrate 2 (FRS2) docking proteins. (novusbio.com)
  • downstream of GPCR activation, including activation via second messenger, immediate phosphorylation or immediate G proteins interaction. (tsfaq.info)
  • They found that ICP0 exploits phosphorylation, a chemical mark that is often used in cells to promote interactions between proteins, especially as part of the cellular signaling response to DNA damage. (infectioncontroltoday.com)
  • The researchers also found that ICP0 exploits the same phosphorylation signal to bind to other cellular proteins in addition to RNF8, a hint that it may play a broader role in defeating antiviral defenses and manipulating cellular machinery. (infectioncontroltoday.com)
  • FGF stimulation leads to phosphorylation of Shp2 on a tyrosine residue forming a complex with an additional molecule of Grb2. (novusbio.com)
  • Grb2/Sos complexes are thus recruited directly and indirectly via Shp2 upon tyrosine phosphorylation of FRS2a in response to growth factor stimulation. (novusbio.com)
  • In the present study, we demonstrated that FAK is implicated in RCP-induced EGFR phosphorylation and ovarian cancer cell invasion with inhibition by curcumin. (nature.com)
  • Inhibition of pRb phosphorylation and cell cycle progression by an antennapedia-p16(INK4A) fusion peptide in pancreatic cancer cells. (semanticscholar.org)
  • The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. (sdu.dk)
  • The study suggests that the mechanical changes caused by inhibition of cellular respiration may involve the combined effect of several metabolic alterations, including decreased LC20 phosphorylation during contraction, but apparently not decreased intracellular Ca2+ concentration or sensitivity of the contractile system to Ca2+. (lu.se)
  • Structural changes in phosphorylated triple helical collagen-like peptides and requirements for in vitro enzymatic phosphorylation. (tufts.edu)
  • Structural characterization and in vitro phosphorylation of short, collagen-like peptides containing phosphorylation sites found in human the human type I alpha-I collagen chain. (tufts.edu)
  • Combined oxidative phosphorylation deficiency 1 is a severe condition that primarily impairs neurological and liver function. (medlineplus.gov)
  • Liver disease is common in people with combined oxidative phosphorylation deficiency 1, with individuals quickly developing liver failure. (medlineplus.gov)
  • Individuals with combined oxidative phosphorylation deficiency 1 usually do not survive past early childhood, although some people live longer. (medlineplus.gov)
  • Combined oxidative phosphorylation deficiency 1 is likely a rare disorder, although its prevalence is unknown. (medlineplus.gov)
  • Combined oxidative phosphorylation deficiency 1 is caused by mutations in the GFM1 gene. (medlineplus.gov)
  • The condition is called combined oxidative phosphorylation deficiency 1 because it impairs the function of more than one of these complexes. (medlineplus.gov)
  • A shortage of energy in these tissues leads to cell death, causing the neurological and liver problems in people with combined oxidative phosphorylation deficiency 1. (medlineplus.gov)
  • Many differences in expression or phosphorylation state were observed for these two embryonic stages. (uu.nl)
  • Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression at the surface of hippocampal neurons. (portlandpress.com)
  • We identified multiple basic residues in the carboxyl terminus of Smo that were required for interaction with PKAc, Smo phosphorylation, and Hh pathway activation. (science.org)
  • R)-alpha-lipoic acid (LA), a dithiol compound with antioxidant properties, is effective against age-related increases in oxidative stress and has been used to improve glucose utilization through insulin receptor (IR) pathway-mediated Akt phosphorylation. (oregonstate.edu)
  • Analyses of the transport cycle show that serine phosphorylation abolishes the K + -dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential. (sdu.dk)
  • Phosphorylation of the serine residues in estrogen receptor (ER) α is important in transcriptional activation. (reading.ac.uk)
  • HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. (garvan.org.au)
  • Thus, our study indicates that gentisic acid promotes the proliferation of keratinocyte by phosphorylation of ERK1/2. (medsci.org)
  • For example, in alkali burn wound recovery, phosphorylation of ERK1/2 has been promoted by the compound body protective compound-157 in human umbilical vein endothelial cells to facilitate proliferation and wound repair [ 13 ]. (medsci.org)
  • Treatment of HEK293 cells (50,000 cells/well) with EGF for 10 min at RT induced the phosphorylation of ERK1/2 at T202/Y204. (bmglabtech.com)
  • iwasakii exhibits cell cycle dysregulation, ERK1/2/p38 MAPK/AKT phosphorylation, and reduced MMP-9-mediated metastatic capacity in prostate cancer models in vitro and in vivo. (bvsalud.org)
  • CE stimulated the phosphorylation of ERK1/2, p38 MAPK , and AKT in DU145 cells , but only p38 MAPK phosphorylation was increased in LNCaP cells . (bvsalud.org)
  • Investigation into the possible mechanism(s) underlying this activation revealed that these membranes contain endogenous systems which prefer to utilize GTP, rather than ATP, in the phosphorylation of specific protein substrates with apparent M.W. of 54K and 33K. (elsevier.com)
  • In particular, phosphorylation of the ERα is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30. (reading.ac.uk)
  • After activation the level of phosphorylation increased to 30 +/- 3% (n = 9) in the control veins. (lu.se)
  • In summary, this study identified a novel binding partner of CYLD and characterised the phosphorylation-mediated activation of its DUB activity. (bl.uk)
  • Epidermal growth factor receptor (EGFR) phosphorylation and activation has been identified as one important mediator of inflammatory cytokine release from these cells. (cdc.gov)
  • TAp73alpha induces tau phosphorylation in HEK293a cells via a transcription-dependent mechanism. (ox.ac.uk)
  • In this pilot study we show by Western blotting that TAp73alpha induces phosphorylation of human 2N4R tau at threonine-205 and at the PHF-1 epitope (serine366/serine404) in HEK293a cells. (ox.ac.uk)
  • Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. (nih.gov)
  • Now the main player, when we're talking about cellular respiration and Oxidative Phosphorylation, is NADH. (khanacademy.org)
  • Taken together this research emphasizes the strength of mass spectrometry based phosphoproteomics in the elucidation of endogenous signaling pathways via the large scale identification of phosphoproteins and phosphorylation sites and the obtained results contribute to a better understanding of biological pathways and critical cellular processes. (uu.nl)
  • Cell cycle-dependent phosphorylation and regulation of cellular differentiation. (cam.ac.uk)
  • We now show that short-term treatment of quiescent Swiss 3T3 cells with RA yields marked increases in uridine phosphorylation and total cellular RNA synthesis as well as 2-deoxyglucose uptake. (aacrjournals.org)
  • Conclusions: These studies extend the evidence for EGFRs as important cellular targets for components of HDE and they reveal novel patterns of EGFR phosphorylation and binding changes that vary among airway epithelial cell types. (cdc.gov)
  • In HSV-1 infection, the phosphorylation signal on ICP0 attracts a cellular DNA damage response protein, RNF8, which binds to the false signaling marker and is then degraded. (infectioncontroltoday.com)
  • Viral E3 Ubiquitin Ligase-Mediated Degradation of a Cellular E3: Viral Mimicry of a Cellular Phosphorylation Mark Targets the RNF8 FHA Domain,' Molecular Cell, published online March 8, 2012, to appear in print, April 13, 2012. (infectioncontroltoday.com)
  • Thus, low availability of the above-mentioned macrominerals would be expected to delay postprandial cellular uptake of glucose, impair phosphorylation, and eventually hinder carbohydrate metabolism and energy production (10). (who.int)
  • High extracellular glucose and TGF- β levels stimulate ACTN4 phosphorylation. (asnjournals.org)
  • Oral treatment of specific commercial Bifidobacterium longum and Bifidobacterium bifidum enhanced bile acid signaling contributing to potentiate oxidative phosphorylation (OXPHOS) in adipose tissues, leading to reduction of body weight gain and improvement in hepatic steatosis and glucose homeostasis. (biomedcentral.com)
  • Recent studies on C6 sugar nucleoside antifungal natural products, such as nikkomycins and polyoxins, revealed a novel biosynthetic mechanism involving cryptic phosphorylation. (duke.edu)
  • Here, we examine the transcription of genes associated with all five mitochondrial and nuclear genome-encoded oxidative phosphorylation (OXPHOS) complexes, the primary physiological mechanism responsible for the production of ATP, in whitefish species pairs from Cliff Lake and Webster Lake in Maine, USA. (datadryad.org)
  • While PTM comprises a vast range of residues and cleavage sites, the phosphorylation of amino acid side chains is by far the most common modification. (bmglabtech.com)
  • Therefore we have explored the possibilities to use zebrafish development as a model system to study in vivo phosphorylation using mass spectrometry. (uu.nl)
  • My studies reveal a novel phosphorylation site (S568) on CYLD which regulates CYLD activity and its ability to restrict innate immune signalling. (bl.uk)
  • Importance of glycolysis and oxidative phosphorylation in advanced melanoma. (harvard.edu)
  • These findings suggest that, in addition to genetic mutations, increased phosphorylation of ACTN4 may mediate podocyte injury and kidney disease. (asnjournals.org)
  • H2A.X Phosphorylation Assay Kit (Flow Cytometry) The H2A.X Phosphorylation Assay Kit (Flow cytometry) is a cell based assay formatted for flow cytometric detection of levels of phosphorylated Histone H2A.X. (sigmaaldrich.com)
  • The H2A.X Phosphorylation Assay Kit (Flow cytometry) is a cell based assay formatted for flow cytometric detection of levels of phosphorylated Histone H2A.X. Cells are cultured in microplates, treated with agents that induce DNA damage or apoptosis, which stimulates H2A.X phosphorylation. (sigmaaldrich.com)
  • Nevertheless, we demonstrate here temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC messenger RNA accumulation under continuous dark conditions. (harvard.edu)
  • We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. (nih.gov)
  • We identified more than 1000 phosphorylation sites in 60 embryos. (uu.nl)
  • Comparison with human datasets revealed surprising conservation of phosphorylation sites, suggesting specialized function. (uu.nl)
  • Heatmap displaying changes in absolute occupancy of individual phosphorylation sites. (xenbase.org)
  • Nine groups were manually selected from the top 25% of sites having the greatest variation in phosphorylation across the seven experimental time points. (xenbase.org)
  • This activity is independent of the identified phosphorylation sites. (portlandpress.com)
  • identify the phosphorylation sites of ARX. (portlandpress.com)
  • Incubation of HeLa cells (50,000 cells/well) with Anisomycin for 60 min at 37°C stimulates phosphorylation of p38αβγ at T180/Y182. (bmglabtech.com)
  • Regulation of RCAN1 protein activity by Dyrk1A protein-mediated phosphorylation. (bvsalud.org)
  • The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. (medlineplus.gov)
  • 2017), Phosphorylation Dynamics Dominate the Regulated. (xenbase.org)
  • Ectopic expression of RCP induced FAK phosphorylation, which links β1 integrin with EGFR and participates in a positive regulation loop with EGFR. (nature.com)
  • In the present study, we showed that FAK is implicated in RCP-induced EGFR phosphorylation, leading to ovarian cancer cell invasion. (nature.com)
  • EGFR phosphorylation, expression, and localization were assessed with anti-EGFR antibodies and either blotting or confocal microscopy. (cdc.gov)
  • HDE stimulated EGFR phosphorylation at both 15 min and 18 hr in BEAS-2B cells and primary cells, but only at 15 min in H292 cells, indicating that the different EGFR binding changes among these cell types is likely related to their different time-dependent changes in phosphorylation. (cdc.gov)
  • Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. (uzh.ch)
  • Phosphorylation mediates high tempera. (ugent.be)
  • L. D. Vu, "Phosphorylation mediates high temperature signalling in plants," Ghent University. (ugent.be)
  • Here we link BRI1-mediated phosphorylation to another growth regulatory network that directly mediates protein stability. (jic.ac.uk)
  • Organs that have high energy demands, such as the brain and liver, are particularly affected by the resulting impairment of oxidative phosphorylation. (medlineplus.gov)
  • This dual monoubiquitylation-phosphorylation regulation supports the key role of BR levels in maintaining the proliferative potential of cells during organ growth. (jic.ac.uk)
  • Upon subsequent treatment of the cells with phorbol myristate acetate, a direct correlation between the comitogenic activity of RA and its stimulation of uridine phosphorylation and RNA synthesis is apparent. (aacrjournals.org)
  • Details] Phosphorylation and gene expression of p53 are not affected in human cells exposed to 2.1425 GHz band CW or W-CDMA modulated radiation allocated to mobile radio base stations [med. (emf-portal.org)
  • Here we show that a phosphoablative substitution in MacP that blocks StkP-mediated phosphorylation prevents PBP2a activity without affecting the MacP-PBP2a interaction. (whiterose.ac.uk)
  • Phosphorylation of the histone variant H2A.X is a rapid and sensitive response to double strand DNA breaks. (sigmaaldrich.com)
  • Mapping protein phosphorylation in zebrafish development Reversible protein phosphorylation plays a key role in signaling processes that are vital for a cell and organism. (uu.nl)
  • A quantitative proteomics approach, using stable isotope di-methyl labeling was used to compare protein phosphorylation between wild type and Fyn/Yes knockdown embryos that display convergence and extension cell movement defects. (uu.nl)
  • We detected differential phosphorylation of known regulators of gastrulation cell movements, including Gravin, PDLIM5 and small GTPase regulators. (uu.nl)
  • Here, we focus on a direct mechanistic link involving phosphorylation of differentiation-associated transcription factors by cell cycle-associated Cyclin-dependent kinases. (cam.ac.uk)
  • Brassinosteroids (BR) have centrally important functions in plant growth by promoting cell proliferation and cell expansion through phosphorylation-mediated regulatory cascades that are initiated by perception of BR by receptor-like kinases of the BRI1 family. (jic.ac.uk)
  • Biphasic changes in airway epithelial cell EGF receptor binding and phosphorylation induced by components of hogbarn dust. (cdc.gov)
  • Using in situ protein phosphorylation analyzed by SDS-PAGE and autoradiography, we observed that B-50 phosphorylation was significantly decreased by 3,4-DAP in the presence of extracellular Ca2+ and completely inhibited by removal of extracellular Ca2+. (chinaphar.com)
  • Details] Mobile phone base station-emitted radiation does not induce phosphorylation of Hsp27 [med. (emf-portal.org)
  • Phosphorylation of the 20-kDa regulatory light chains (LC20) of the myosin molecule was studied in the relaxed state and at the plateau of high-K+ contractures for comparison with the mechanical data. (lu.se)
  • Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion. (strath.ac.uk)
  • A protein expressed by yeast system could be modificated such as glycosylation, acylation, phosphorylation and so on to ensure the native protein conformation. (antikoerper-online.de)
  • The results established zebrafish as a good model to study in vivo phosphorylation and reveal that surprising homology exists with human phosphoproteins. (uu.nl)
  • In this thesis in vivo phosphorylation was studied at different stages and under different experimental conditions in developing zebrafish embryos. (uu.nl)
  • To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. (tubitak.gov.tr)
  • Los experimentos in Vivo de Lai y Singh (1995, 1996) ameritan especial atención, teniendo en cuenta el interés que despertaron. (rfcom.ca)
  • The earliest stages of animal development are largely controlled by changes in protein phosphorylation mediated by signaling pathways and cyclin-dependent kinases. (xenbase.org)
  • K in its turn acts as a cofactor for several enzymes involved in carbohydrate phosphorylation and oxidation, such as protein kinases and phosphatases. (who.int)
  • IP7 phosphorylation appears selective for eukaryotic organisms, with none seen in Escherichia coli extracts. (the-scientist.com)
  • We found an age-related lesion in Akt activity via loss of phosphorylation on Ser473. (oregonstate.edu)
  • Neither the dominant negative isoform, DeltaNp73, nor a transcriptionally inactive mutant TAp73alpha(R292H) altered tau phosphorylation indicating that tau phosphorylation is dependent on the transcriptional activity of TAp73alpha. (ox.ac.uk)
  • In the second part of the thesis I investigate the regulation of CYLD's catalytic activity by phosphorylation. (bl.uk)
  • Therefore, dual biomarkers of BChE activity and phosphorylation (OP-BChE) will be obtained simultaneously. (cdc.gov)
  • As a consequence protein phosphorylation and the elucidation of signaling pathways have received considerable attention over the past years. (uu.nl)
  • Erythrocyte membrane phosphorylation in Plasmodium berghei infected mice / by Porntip Chaimanee. (who.int)
  • These BR-mediated growth responses have been explained by transcriptional controls mediated by phosphorylation of BES1/BZR1 transcription factors. (jic.ac.uk)