(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...
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 - 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 ...
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 ...
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 ...
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 ...
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 ...
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 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 ...
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 ...
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 ...
Clone REA134 recognizes AKT1, which is also known as protein kinase Bα. AKT1 is a serine/threonine protein kinase, belonging to the AKT family of kinases and like each AKT family member, contains an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a carboxyl-terminal regulatory domain with a hydrophobic motif (HM). Activation of AKT1 is achieved via phosphorylation at multiple sites, which take place in response to engagement of receptors such as platelet derived growth factor receptor (PDGF-R). Activated AKT1 further phosphorylates and alters the activity of several downstream substrates allowing AKT1 to play a vital role in various biological processes such as cell growth, survival, migration, and proliferation. Additional information: Clone REA134 displays negligible binding to Fc receptors. - Belgique
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If you have an antibody against the protein you investigate you can perform 1D IEF gels followed by western blots (see my current post). The blotting effiency is not very high because the proteins in the gel are not charged but I try to optimize (washing with SDS). With this method you can detect different phosphorylated forms of that protein. And if you have a suggestion about the respective kinase and are able to construct a mutant (Im working with bacteria...) the phosphorylated band should disapear if your suggestion is right ...
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T-lymphocytes play a crucial role in the immune response, both as direct effector cells and as regulatory cells that modulate the functions of other cell types. One of the earliest recognizable events after TCR stimulation is activation of LCK (p56lck) and FYN (p59fyn), which results in phosphorylation of tyrosine residues. Subsequently, ZAP70 tyrosine kinase is recruited to the TCR where it is activated by LCK through tyrosine phosphorylation. ZAP70 is a key signaling molecule in T cells, where it couples the antigen-activated TCR to downstream signaling pathways.28 Once ZAP70 has been activated, LCK and ZAP70 presumably act synergistically to phosphorylate specific downstream substrates, which in turn orchestrate the cytoplasmic signaling cascades leading to T-cell activation. These central roles of LCK and ZAP70 in TCR signaling have been amply documented. ZAP70 causes phosphorylation of LAT (linker for activation of T-cells), which in turn activates important downstream signaling pathways, ...
This chapter focuses on other modalities of signal transduction, including intracellular second messengers, feedback regulation, and posttranslational modifications such as the phosphorylation-dephosphorylation of proteins. Empirical studies of archaeal-archaeal and archaeal-bacterial communication have been few in number and preliminary in nature. Inspection of archaeal genomes has revealed them to be devoid of homologs of the prototypic bacterial quorum-sensing proteins LuxS and LuxR. Two-component systems differ in several fundamental respects from protein-serine/threonine/tyrosine phosphorylation cascades. First, autophosohorylation is the predominant mechanism of phosphorylation in the two-component system, whereas protein-serine/threonine/tyrosine phosphorylation cascades rely primarily on phosphotransfer reactions catalyzed by protein kinases that are distinct from the phosphoacceptor protein. Second, the chemical nature of the phosphoryl moieties formed during two-component signaling differs
Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulin receptor substrate-1 (IRS-1). While the pathway responsible for IRS-1 degradation in this system is unknown, c-Jun NH2-terminal kinase (JNK) has been linked with serine phosphorylation of IRS-1 and insulin resistance. We investigated the role of JNK in ANG II-induced IRS-1 phosphorylation, degradation, Akt activation, glucose uptake, and hypertrophic signaling, focusing on three IRS-1 phosphorylation sites: Ser302, Ser307, and Ser632. Maximal IRS-1 phosphorylation on Ser632 occurred at 5 min, on Ser307 at 30 min, and on Ser302 at 60 min. The JNK inhibitor SP600125 reduced ANG II-induced IRS-1 Ser307 phosphorylation (by 80%), IRS-1 Ser302 phosphorylation (by 70%), and IRS-1 Ser632 phosphorylation (by 50%). However, JNK inhibition had no effect on ANG II-mediated IRS-1 degradation, nor did it reverse the ...
Angiotensin II (ANG II) has been implicated in the pathogenesis of diabetic micro- and macrovascular disease. In vascular smooth muscle cells (VSMCs), ANG II phosphorylates and degrades insulin receptor substrate-1 (IRS-1). While the pathway responsible for IRS-1 degradation in this system is unknown, c-Jun NH2-terminal kinase (JNK) has been linked with serine phosphorylation of IRS-1 and insulin resistance. We investigated the role of JNK in ANG II-induced IRS-1 phosphorylation, degradation, Akt activation, glucose uptake, and hypertrophic signaling, focusing on three IRS-1 phosphorylation sites: Ser302, Ser307, and Ser632. Maximal IRS-1 phosphorylation on Ser632 occurred at 5 min, on Ser307 at 30 min, and on Ser302 at 60 min. The JNK inhibitor SP600125 reduced ANG II-induced IRS-1 Ser307 phosphorylation (by 80%), IRS-1 Ser302 phosphorylation (by 70%), and IRS-1 Ser632 phosphorylation (by 50%). However, JNK inhibition had no effect on ANG II-mediated IRS-1 degradation, nor did it reverse the ...
Gang Zhang from the Nilsson group has been investigating the spindle assembly checkpoint in human cells and how it controls the timing of chromosome segregation. In particularly he has focused on understanding how the checkpoint protein Mad1 localized to kinetochores, large protein structures on chromosomes that bind to microtubules. It is known that Mad1 localization is important for checkpoint function but the underlying protein-protein interactions and their regulation have not been uncovered.. In collaboration with the Nielsen lab at CPR the researchers used an in vivo proximity dependent biotinylation approach coupled with mass spectrometry to identify Mad1 interactors. This revealed a so far elusive interaction with the Bub1 checkpoint protein that the researchers further characterized. Interestingly the binding of Mad1 and Bub1 required the phosphorylation of Bub1 by mitotic kinases and a novel generated phospho-specific antibody revealed that Bub1 was specifically phosphorylated on ...
Detection of phosphorylation by western blotting is an important procedure to elucidate molecular mechanisms in signal transduction pathways involving kinases and phosphatases. Anti-phospho-tyrosine monoclonal antibodies have been widely used because they react with plethora of proteins containing phosphorylated tyrosine residues. In contrast, the monoclonal antibodies against phospho-serine or threonine residues are unpopular, since their affinity and specificity are less than optimal. To achieve precise characterization of signaling events, it is desirable to raise a good anti-phospho-site-specific antibody to clearly detect phosphorylated species. However, raising this type of antibody is costly and time-consuming, and sometimes results in failure.. Use of Phos-tag may provide an alternative method to detect phosphorylated proteins. Phos-tag is a dinuclear metal complex that acts as a novel phosphate-binding tag. Phos-tag molecules preferentially capture phosphomonoester dianions bound to ...
Phosphorylation and de-phosphorylation play critical roles as a mode of signal transfer in biological processes. Check out our phosphorylation webpage to learn about this process, the proteins being phosphorylated, and the products offered at BioLegend to help you investigate this phenomenon. BioLegend develops and manufactures world-class, cutting-edge immunological reagents for biomedical research, offered at an outstanding value.
It has already been pointed out that a bivalent/multivalent TCR might improve the high off rate found for monovalent MHCp binding and can resolve the paradox of the high specificity-low affinity TCR-MHCp interaction (32). However, this does not readily explain how T cells manage to respond to a wide range of MHCp concentrations (6, 23-25). The coexistence of monovalent and multivalent TCRs may clarify this issue. Theoretical calculations suggest that cells coexpressing monovalent and multivalent forms of a given receptor can show high sensitivity to low concentrations of a ligand and concentration-dependent responses to high ligand concentrations (33). Indeed, by analyzing antigen-stimulated TCRs in BN-PAGE, we show that the multivalent TCR might facilitate signaling at low MHCp doses because only these complexes are phosphorylated at low peptide concentrations. On the other hand, the monovalent TCRs are only phosphorylated at high concentrations of peptide antigen, a situation in which the ...
PTMs (posttranslational modifications) such as ubiquitylation, sumoylation, acetylation and protein methylation are pivotal modifiers that determine the activation, deactivation or subcellular localization of signaling proteins, facilitating the initiation, amplification and transduction of signaling. Accumulating evidence suggest that several key signaling molecules in Hippo signaling pathway are tightly regulated by various types of PTMs. Malfunction of these critical signaling modules such as YAP/TAZ, MAT1/2 and LATS1/2 due to deregulated PTMs has been linked to a variety of human diseases such as cancer. In this review article, we summarized the current understanding of the impact of PTMs in regulating Hippo signaling pathway and further discussed the potential therapeutic intervention from the view of PTMs and Hippo pathway.
Concepts regarding the controls and consequences of PKD1-Ser738/Ser742 (activation loop) phosphorylation are based largely on early studies that used an anti-PKD1-Ser(P)738/Ser(P)742 PSSA (from Cell Signaling Technology, Danvers, MA) and showed that PMA increases PKD1 activation loop phosphorylation in many cell types via a mechanism that requires nPKC isoform activity (PKCδ, PKCε, PKCη, and/or PKCθ). In vitro kinase assays showing direct phosphorylation of the PKD1 activation loop by certain nPKC isoforms also have been published (Brändlin et al., 2002). However, there is recent evidence that the Cell Signaling Technology anti-PKD1-Ser(P)738/Ser(P)742 PSSA primarily recognizes PKD1 phosphorylation at Ser738 and that PKD1 phosphorylation at Ser742 can be tracked with a different PSSA (commercially available from Abcam Inc., Cambridge, MA). Experiments that use a combined approach with these two PSSAs expose differences in the controls and consequences of PKD1 phosphorylation at Ser738 and ...
In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites with central roles in TCR signaling. The model was used to generate predictions suggesting unexpected roles ...
The NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins. Activation of IκBα occurs through both serine and tyrosine phosphorylation events. Activation through phosphorylation at Ser-32 and Ser-36 is followed by proteasome-mediated degradation, resul
In article ,36cp86$3vu at agate.berkeley.edu, lhom at OCF.Berkeley.EDU (Louis Hom) writes: ,,I know antibodies are glycosylated, but are they phosphorylated? ,,-- ,Yes they are. I found that you the hard way while doing in vitro ,phosphorylation experiments :-( , ,Annette ,ah690549 at mbcr.bcm.tmc.edu I do not think they are normally phophorylated. But during in vitro phosphorylation experiments basically every protein around that has serine, threonine or tyrosine gets phosphorylated. So much for kinase specificity. Its obvious that in non physiological conditions (ie. kinase assay on IPs) the specificity goes down the drain ---------------------------------------------------------------------- Michele Fuortes, MD/PhD Dept. Cell Biology and Anatomy Cornell University Medical College 1300 York Ave - Box 57 New York, NY 10021 E-mail: mfuortes at med.cornell.edu ...
Last updated: May 12, 2010. Introduction:. Protein phosphorylation is the most ubiquitous post-translational modification (PTM), and plays important roles in most of biological processes. Identification of site-specific phosphorylated substrates is fundamental for understanding the molecular mechanisms of phosphorylation. Besides experimental approaches, prediction of potential candidates with computational methods has also attracted great attention for its convenience and fast-speed. In this review, we present a comprehensive but brief summarization of computational resources of protein phosphorylation, including phosphorylation databases, prediction of non-specific or organism-specific phosphorylation sites, prediction of kinase-specific phosphorylation sites or phospho-binding motifs, and other tools. A testing data set prepared from Phospho.ELM 6.0 is available at: Comparison_data. We apologized that the computational studies without any web links of databases or tools will not be included ...
Generation of Phospho-Ser65 Parkin and Phospho-Thr257 PINK1 Pre-clinical Monoclonal Antibodies and Characterization of Total PINK1 Pre-clinical Monoclonal ...
Phosphoinositide 3-Kinase (PI3K) is a central enzyme in a signaling pathway that mediates cellular responses to growth factors. This enzyme phosphorylates the 3 position of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. A number of signaling proteins, including the Ser/Thr protein kinases, AKT and PDK1, contain pleckstrin homology domains that bind specifically to PIP3. Thus, the generation of PIP3 at the plasma membrane in response to activation of PI3K by growth factors results in the initiation of downstream Ser/Thr phosphorylation cascades that control a variety of cellular responses. The signaling pathway downstream of PI3K is highly conserved from worms and flies to humans and genetic analysis of the pathway has revealed a conserved role in regulating glucose metabolism and cell growth. Based on deletion of genes encoding the catalytic or regulatory subunits of PI3K in the mouse, PI3K mediates insulin dependent ...
The large number of protein consensus sequences that may be recognized without computer analysis are reviewed. These include the extensive range of known phosphorylation site motifs for protein kinase
The chapters have been organized into 5 different sections, based on the similarity of protein phosphorylation pathways. The first section has 4 chapters summarizing on the role of Akt, mTOR and AMPK in cancer and metabolic disorders. The section focuses on the structure, function and regulation of mTOR1 and mTOR2 and their role in cancer, neurodegeneration, and diabetes, the role of Sestrins, a family of stressresponsive genes in regulating AMPK-mTOR pathway and their role in cardiovascular diseases, muscle- and neuro- degeneration, diabetes and cancer, the signaling pathways that regulate autophagy, including Akt, AMPK and mTOR, and the role of Akt/AMPK and PI3K/Erk pathways in fatty acids-stimulated glucose uptake. The second section has 4 chapters reviewing the role of protein phosphorylation in transcription, pre-mRNA splicing and DNA damage. The section emphasizes the regulation of RNA polymerase II sequentially by protein kinases and phosphatases in gene expression and therapeutically ...
Takaoka et al. have presented persuasive evidence that in MEFs the IFN-γ response is substantially augmented through autocrine IFN-α/β and that cross-recruitment and phosphorylation of the IFNAR1 subunit of the IFN-α/β receptor occurs in response to IFN-γ in these cells (24). We have been unable to obtain evidence for or against recruitment or phosphorylation of IFNAR1 in response to IFN-γ in the HT1080-based human cell systems used here (H. Isharc and I. M. Kerr, unpublished data). In a potentially analogous but inverse situation, cross-phosphorylation of the IFNGR1 subunit of the IFN-γ receptor in response to activation of erythropoietin/gp130 receptor chimeras in HT1080-based cell lines is observed. Importantly, however, such receptor cross-phosphorylation plays no part in the IFN-γ-like response observed (23), a result which emphasizes that even if IFNAR1 were recruited and phosphorylated in response to IFN-γ, proof of necessity of this for the IFN-γ antiviral response would ...
A-Kinase (cAMP dPK). A-Kinase (cAMP dPK),. Cyclic AMP dependent protein kinase. A family of enzymes activated by cyclic AMP, which catalyse intracellular phosphorylation reactions.. Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events.. ...
BioAssay record AID 1078274 submitted by ChEMBL: Inhibition of human full length PKC zeta expressed in baculovirus assessed as substrate phosphorylation using fluorescence-labelled peptides as substrate at 0.2 uM after 90 mins by microfluidic peptide phosphorylation assay.
BioAssay record AID 1079124 submitted by ChEMBL: Inhibition of human full length PKC beta2 expressed in baculovirus assessed as substrate phosphorylation using fluorescence-labelled peptides as substrate at 420 uM after 90 mins by microfluidic peptide phosphorylation assay.
Predicted two-dimensional H2AX phosphorylation patterns with and without the 3D spreading mechanism.Example of XY projections of hit domains only (A) and of all
This is a different type of regulation from the more rapid (de)phosphorylation mechanism, but is by no means unusual in metabolic systems. The actual mechanism of this insulin-initiated regulation is beyond the scope of this course. However, recall that glucokinase is the "high Km" kinase for glucose, so there is really no reason for it to even be around if glucose concentrations stay low most of the time. ...
... : Main EGFR signaling pathways in wound healing and cancer. EGFR occupation by EGF or other cognate agonistic ligand triggers a conformational change within the receptor s topography leading to carboxy-terminal tyrosines phosphorylation and accessory proteins recruitment. Three major signaling pathways have been described upon EGFR occupation. PI3K, phosphatidil inositol 3-kinase, involved in cyto-protection and cell tolerance to hypoxia. PI3K phosphorylates downstream substrates as Akt or PKB on serine 473. Consequently Akt inhibits apoptosis via BAD and BAX inactivation. This pathway assists in cell survival and appears to be involved in wound bed and tumor cells survival when angiogenesis is not accomplished, thus contributing to tumor metastasis. Cell proliferation involves the RAS-RAF-MAPK pathway, where phosphorylated EGFR recruits accessory proteins which activate the oncogene derived proteins RAS, subsequently RAF, and the Mitogen-Activated Protein Kinase (MAPK) pathway leading ...
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 ...
Adenosine triphosphate, labeled on the gamma phosphate group with 35S (ATP?S), is used in protein kinase assays (thiosphosphorylation) in place of ATP. It can be used as a substrate for many protein kinases. One of the advantages of this substrate and its phosphorylated products is that they are hydrolyzed slowly by phosphatases and ATPases, and are therefore more metabolically stable.. ...
This protein phosphorylation antibody pair set comes with two antibodies, one against the TP53 protein, and the other against the specific S9 phosphorylated site of TP53 for use in in situ Proximity Ligation Assay. See Publication Reference below. (DP0014) - Products - Abnova
This protein phosphorylation antibody pair set comes with two antibodies, one against the TP53 protein, and the other against the specific S20 phosphorylated site of TP53 for use in in situ Proximity Ligation Assay. See Publication Reference below. (DP0087) - Products - Abnova
Gong J, Holewinski RJ, Van Eyk JE, Steinberg SF (2016) A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ. Biochem J 473, 311-20 ...
In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein...
It kind of depends on what all is known so far and some of it is just going to be trying and seeing what works. If you know the kinase that phosphorylates the protein you can do in vitro and/or in cyto phosphorylation and seeing if it is activated. If you know the site that is likely phosphorylated you can mutate the residue(s) to alanine(s), which cannot be phosphorylated. You can also do the reverse and mutate the residue(s) to aspartic acid, which have a negative charge and can sometimes mimic the phosphorylated state. It just depends on what you know, what you need to know, and what you have available to you.. ...
The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocyto
Function: Positive effector of BCR-stimulated responses. Couples the B-cell antigen receptor (BCR) to the mobilization of calcium ion either through a phosphoinositide 3-kinase-dependent pathway, when not phosphorylated on tyrosines of the linker region, or through a phospholipase C-gamma-dependent pathway, when phosphorylated on Tyr-348 and Tyr-352. Thus the differential phosphorylation of Syk can determine the pathway by which BCR is coupled to the regulation of intracellular calcium ion (By similarity ...
KEYWORD: 3D-structure Alternative splicing Glycoprotein Immune response Immunoglobulin domain Phosphorylation Signal T-cell Transmembrane ...
In our previous work, we showed by in vitro ARE-specific decay assays that BRF1 activity was regulated at Ser92 by PKB. However, the in vivo experiments reported here reveal that the mechanism is more complex in intact cells in that the PKB-dependent regulation of BRF1 activity could not be evaded by a Ser92 mutation (Fig. 1B), calling for the existence of at least one additional PKB regulatory site on BRF1. This is in keeping with the fact that the Ser92-based mechanism involves binding to 14-3-3, which usually binds to its target proteins at two separate locations (1, 49). We focused on Ser203 as a candidate for the additional phosphoregulatory site because of its homology to TTP Ser178, which serves as one of two 14-3-3 binding sites (21). Although TTP Ser178 is phosphorylated by MK2 (21, 42), we found no evidence that BRF1 Ser203 is phosphorylated in vitro by this kinase or by p38 or ERK1. Instead, the site is strongly phosphorylated by PKB (Fig. 2). Taken together with the in vivo data, ...
reacts with Akt when phosphorylated at Ser473; also reacts with Akt2 and Akt3 when phosphorylated at corresponding residues. Does not recognize Akt phosphorylated at other sites, nor does it recognize phosphorylated forms of related kinases such as PKC or p70 S6 ...
STAT-6 is a central mediator of IL-4-induced gene responses. It has been shown that STAT-6 is expressed in B cells and T cells upon stimulation with IL-4. Interleukin-12 (IL-12) and IL-18 are the key factors for the induction of Th1 Cells, early signals being involved in Th2 differentiation if any by IL-18 are less well characterized. We have investigatedthe mechanisms employed by IL-18 and IL-4 to control expression of STATs. Since we have shown in earlier studies that Th2 cytokines protect cells from apoptosis, we explored the possible role of the controversial cytokine IL-18 on Th2 shift and analyzed the effects of IL-18 on CD4+ T cells. Cells were cultured either with IL-18, IL-4, IL-18 + CD28 for different time points. The optimum time required for the phosphorylation of STAT-6 when stimulated with IL-18 was 72 hours. This effect of STAT-6 phosphorylation was blocked by addition of anti-IL-18 antibody in the culture. However, phosphorylation of STAT-1 and STAT-4 was seen by 24 hours. ...
Zhou J (2012) CST Curation Set: 14185; Year: 2012; Biosample/Treatment: cell line, Jurkat/calyculin_A & pervanadate; Disease: T cell leukemia; SILAC: -; Specificities of Antibodies Used to Purify Peptides prior to LCMS: p[STY] Antibodies Used to Purify Peptides prior to LCMS: Phospho-Tyrosine (P-Tyr-1000) Rabbit mAb Cat#: 8954, Phospho-Tyrosine Rabbit mAb (p-Tyr-1000) Immunoaffinity Beads Cat#: 8876 ...
Creative Bioarray offers a wide range of cell line testing and assays from cell viability and proliferation to cellular phosphorylation assays.
Protein phosphorylation significantly impacts protein function. Find products, protocols, articles and pathways for protein phosphorylation research.
Mouse Monoclonal Anti-Phospho-Tyrosine Antibody (G104) [DyLight 350]. Validated: WB, ICC/IF, IHC, IHC-P. Tested Reactivity: Non-species specific. 100% Guaranteed.
Acts as an adapter for the receptor ERBB2, in epithelia. By binding the unphosphorylated ERBB2 Tyr-1248 receptor, it may contribute to stabilize this unphosphorylated state (By similarity). Inhibits NOD2-dependent NF-kappa-B signaling and proinflammatory cytokine secretion (PubMed:16203728).
We are interested in how various bacteria (e.g., the model Gram+ spore-former B. subtilis and S. aureus) enter in dormancy and also re-initiate growth. We have identified several well-conserved, eukaryotic-like Ser/Thr kinase that are essential for these processes and are investigating the mechanism underlying their function. Among their targets are two essential GTPases, EF-Tu and EF-G, that are key in regulating protein synthesis. Specifically, we are examining how this phosphorylation changes the activity of these protein. In addition, we are examining the role of this kinase in antibiotic resistance and survival during stationary phase ...
Phosphatidylinositol (PtdIns) and its phosphorylated derivatives represent less than 5% of total membrane phospholipids in cells. Despite their low abundance, they form a dynamic signalling system that is regulated in response to a variety of extra and (...). ...
Loss of eif-2alpha phosphorylation on S49 (mammalian S51) associated with the integrated stress response hastens development in C. elegans ...
How is Phosphorylated 112 kDa Protein abbreviated? p112 stands for Phosphorylated 112 kDa Protein. p112 is defined as Phosphorylated 112 kDa Protein very rarely.
ProSci offers more than 700 cell lysates that have been rigorously tested for your research projects. Order your research lysates online from ProSci today.
ProSci offers more than 700 cell lysates that have been rigorously tested for your research projects. Order your research lysates online from ProSci today.
This overview provides a history of protein phosphorylation research and provides the reader with an understanding of how and why labeling studies are performed
Gentaur molecular products has all kinds of products like :search , Gene Link \ Not I non_phosphorylated linker \ 26-3200-14 for more molecular products just contact us
neurofilament-associated kinase: phosphorylates a subset of peptides in vitro which are phosphorylated in vivo in cultured neurons
... analysis of phosphorylated proteins allows researchers a quick and effective way to measure signalling cascades in individual cells.
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. ...
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 ...