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.