Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for renaturing and detecting the activity of protein tyrosine phosphatases (PTPases) after sodium dodecyl sulfate (SDS)-gel electrophoresis with greatly improved sensitivity and resolution is described. Epidermal growth factor receptor-kinase, c-src kinase, and focal adhesion kinase were phosphorylated on tyrosine with 32PO4 and incorporated into gels prior to electrophoresis. These proteins are dephosphorylated when cellular proteins are electrophoresed and the separated PTPases are renatured in the gel by removing SDS with extensive washing. With whole cell lysates, at least eight separate bands of decreased radioactivity corresponding to PTPase activity with molecular weights between 110 and 34 kDa are seen in autoradiographs of the dried gels. PTPases detected are similar with different cell types and with the three 32P-labeled protein substrates, although they are different in cytosolic and membrane-associated fractions. A PTPase detected above 200 kDa in wheat germ agglutinin eluates from solubilized cells suggests that some receptor PTPases can be renatured. While microgram levels of recombinant PTP-1C are required for detection, nanogram levels of recombinant PTP-1B are easily detected. Assaying the activity of renatured PTPases after they have been separated by molecular weight in SDS gel electrophoresis provides a simple and rapid means of determining the activity of individual PTPases in cell fractions.
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PMID:Activity and molecular weight of protein tyrosine phosphatases in cell lysates determined by renaturation after gel electrophoresis. 866 May 68

The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin alphaIIbbeta3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin alphaIIbbeta3) and signaling enzymes (pp60c-src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin alphaIIbbeta3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin alphaIIbbeta3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50-80% reduction in the amount of cytoskeletal associated talin, integrin alphaIIbbeta3, PI 3-kinase, FAK, pp60(c-)src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin alphaIIbbeta3 in vWf-induced signal transduction.
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PMID:Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction. 926 16

Recent studies have focused attention on the role of protein tyrosine kinases in vascular smooth muscle cell biology, but similar information regarding protein tyrosine phosphatases (PTP) is sparse. PTP-1B is a ubiquitous nonreceptor phosphatase with uncertain function and substrates that are mostly unidentified. We used antisense oligodeoxynucleotides (ODN) against PTP-1B to investigate the role of endogenous PTP-1B in motility of primary cultures of rat aortic smooth muscle cells (RASMC). Antisense ODN decreased PTP-1B protein levels and activity in a concentration-dependent fashion, whereas sense, scrambled, or three-base mismatch antisense ODN had little or no effect. Treatment of cells with antisense ODN, but not sense, scrambled, or three-base mismatch antisense ODN, enhanced cell motility and increased tyrosine phosphorylation levels of focal adhesion proteins paxillin, p130(cas), and focal adhesion kinase. Our findings indicate that PTP-1B is a negative regulator of RASMC motility via modulation of phosphotyrosine levels in several focal adhesion proteins and suggest the involvement of PTP-1B in events such as atherosclerosis and restenosis, which are associated with increased vascular smooth muscle cell motility.
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PMID:Role of PTP-1B in aortic smooth muscle cell motility and tyrosine phosphorylation of focal adhesion proteins. 1040 97

Related adhesion focal tyrosine kinase (RAFTK) (also known as PYK2) is a cytoplasmic tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). RAFTK is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic acid, and bradykinin. Overexpression of RAFTK induces activation of c-Jun amino-terminal kinase (also known as stress-activated protein kinase), mitogen-activated protein kinase (MAPK), and p38 MAPK. The present studies demonstrate that RAFTK binds constitutively to the protein tyrosine phosphatase SHPTP1. In contrast to PTP1B, overexpression of wild-type SHPTP1 blocks tyrosine phosphorylation of RAFTK. The results further demonstrate that RAFTK is a direct substrate of SHPTP1 in vitro. Moreover, treatment of PC12 cells with bradykinin is associated with inhibition in tyrosine phosphorylation of RAFTK in the presence of SHPTP1. Furthermore, in contrast to the phosphatase-dead SHPTP1 C453S mutant, overexpression of wild-type SHPTP1 blocks interaction of RAFTK with the SH2-domain of c-Src and inhibits RAFTK-mediated MAPK activation. Significantly, cotransfection of RAFTK with SHPTP1 did not inhibit RAFTK-mediated c-Jun amino-terminal kinase activation. Taken together, these findings suggest that SHPTP1 plays a negative role in PYK2/RAFTK signaling by dephosphorylating RAFTK.
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PMID:Negative regulation of PYK2/related adhesion focal tyrosine kinase signal transduction by hematopoietic tyrosine phosphatase SHPTP1. 1052 52

The molecular mechanism whereby tumor necrosis factor-alpha (TNF-alpha) induces insulin resistance in obesity is not well understood. Previously, we have shown that inhibition of TNF-alpha improved hepatic insulin sensitivity in obese Zucker rats without altering the tyrosine phosphorylation of liver insulin receptors (IRs), which indicates that the TNF-alpha and insulin-signaling cascades interact distally to the IR. To assess the effects of TNF-alpha on signaling molecules downstream from the IR, we analyzed the tyrosine phosphorylation patterns of liver homogenate proteins from TNF-alpha-neutralized fa/fa rats and showed that focal adhesion kinase (FAK) was consistently hyperphosphorylated (4.5-fold). Moreover, intravenous insulin increased hepatic FAK phosphorylation in a time-dependent manner in Sprague-Dawley rats, which suggests that TNF-alpha may induce hepatic insulin resistance by preventing FAK phosphorylation in response to insulin treatment. To explore the cellular mechanism whereby TNF-alpha regulates phosphorylation of FAK in the liver, we measured c-Src kinase activity and the abundance of 3 major protein tyrosine phosphatases (PTPs) (PTP-1B, leukocyte antigen-related tyrosine phosphatase [LAR], and src homology 2 domain-containing protein-tyrosine phosphatase [SHPTP-2]) in liver homogenates from obese Zucker rats after TNF-alpha blockade. Hepatic c-Src kinase activity was unaltered, but LAR protein was reduced by 75%. In addition, TNF-alpha blockade reduced hepatic PTP activity toward tyrosine phosphorylated FAK by 70%, and this was accounted for by immunodepletion of LAR. Incubation of HepG2 cells with TNF-alpha increased LAR protein levels in a dose-dependent manner. Additionally, pretreatment with TNF-alpha abolished insulin-stimulated tyrosine phosphorylation of FAK in HepG2 cells but had no effect on IR tyrosine phosphorylation or expression. These data suggest that TNF-alpha promotes LAR expression and thus prevents insulin-mediated tyrosine phosphorylation of FAK. This probably represents the interface between TNF-alpha and insulin signaling in the liver.
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PMID:Tumor necrosis factor-alpha induces hepatic insulin resistance in obese Zucker (fa/fa) rats via interaction of leukocyte antigen-related tyrosine phosphatase with focal adhesion kinase. 1090 91

Prolactin (PRL) plays a central and crucial role in the regulation of milk protein gene expression in mammary epithelial cells. PRL binding to its cognate receptor leads to receptor dimerization and activation of the tyrosine kinase Janus kinase 2 (JAK2), associated with the membrane-proximal, intracellular domain of the receptor. In turn, JAK2 phosphorylates and activates STAT5, a member of the signal transducers and activators of transcription (STAT) family. We have recently reported that 16 different protein-tyrosine phosphatases (PTP) were expressed in lactating mouse mammary gland and mammary epithelial cells (Aoki, N., Kawamura, M., Yamaguchi-Aoki, Y., Ohira, S., and Matsuda, T. (1999) J. Biochem. (Tokyo) 125, 669-675). We investigated the involvement of each PTP in PRL signaling. Among the 12 phosphatases including SHP-2 examined, a cytosolic phosphatase PTP1B was found to specifically dephosphorylate STAT5a and STAT5b in transfected COS7 and in vitro. Nuclear translocation of STAT5a and STAT5b was largely inhibited upon overexpression of PTP1B. The PRL-dependent transcriptional activation of the beta-casein gene promoter was also inhibited by PTP1B. Furthermore, retrovirus-mediated overexpression of PTP1B resulted in dephosphorylation of endogenous STAT5 and down-regulation of beta-casein gene expression in mammary epithelial COMMA-1D cells when the cells were treated with lactogenic hormones. Endogenous tyrosine-phosphorylated STAT5 proteins in mammary epithelial COMMA-1D cells as well as tyrosine-phosphorylated STAT5a and STAT5b expressed in COS7 cells were co-precipitated by substrate-trapping mutants of recombinant PTP1B. These results strongly suggest that PTP1B dephosphorylates PRL-activated STAT5a and STAT5b, thereby negatively regulating PRL-mediated signaling pathway.
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PMID:A cytosolic protein-tyrosine phosphatase PTP1B specifically dephosphorylates and deactivates prolactin-activated STAT5a and STAT5b. 2158 78

The reversible tyrosine phosphorylation of proteins, modulated by the coordinated actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs), regulates the cellular response to a wide variety of stimuli. It is established that protein kinases possess discrete sets of substrates and that substrate recognition is often dictated by the presence of consensus phosphorylation sites. Here, we have extended this concept to the PTPs and demonstrated that (E/D)-pY-pY-(R/K) is a consensus substrate recognition motif for PTP1B. We have shown that JAK2 and TYK2 are substrates of PTP1B and that the substrate recognition site within theses kinases is similar to the site of dephosphorylation previously identified within the insulin receptor. A substrate-trapping mutant of PTP1B formed a stable interaction with JAK2 and TYK2 in response to interferon stimulation. Expression of wild type or substrate-trapping mutant PTP1B inhibited interferon-dependent transcriptional activation. Finally, mouse embryo fibroblasts deficient in PTP1B displayed subtle changes in tyrosine phosphorylation, including hyperphosphorylation of JAK2. The closely related JAK family member, JAK1, which does not match the consensus dephosphorylation site, was not recognized as a substrate. These data illustrate that PTP1B may be an important physiological regulator of cytokine signaling and that it may be possible to derive consensus substrate recognition motifs for other members of the PTP family, which may then be used to predict novel physiological substrates.
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PMID:TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B. 1169 1

Erythropoietin (EPO) is the principal hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Binding of ligand to the cell-surface EPO-R (EPO receptor) induces dimerization and JAK2 (Janus kinase 2)-mediated tyrosine phosphorylation of the receptor. Less than 1% of the EPO-Rs are displayed on the cell surface; most of the receptor molecules are retained in intracellular compartments, including the ER (endoplasmic reticulum). Using pervanadate (PV) as a potent tool to inhibit cellular PTPs (protein tyrosine phosphatases), we demonstrated previously the accumulation of mature (endoglycosidase H-resistant) tyrosine-phosphorylated EPO-R [Cohen, Altaratz, Zick, Klingmuller and Neumann (1997) Biochem. J. 327, 391-397]. In the present study, we investigated the participation of the ER-associated PTP1B in the dephosphorylation of intracellular EPO-R. We demonstrate tyrosine phosphorylation of EPO-R in BOSC-23T cells co-expressing EPO-R and the 'substrate-trapping' mutant form of PTP1B, PTP1B D181A (referred to as PTP1BD). In vivo interaction between EPO-R and PTP1B suggested that PTP1B dephosphorylates the EPO-R intracellularly. Endoglycosidase H resistance of tyrosine-phosphorylated EPO-R in cells expressing PTP1BD suggested that mature EPO-R is dephosphorylated by PTP1B. Stimulation with EPO of cells co-expressing EPO-R and either PTP1BD or PTP1B resulted in an increase or decrease respectively in phosphotyrosine EPO-R. We thus suggest that PTP1B dephosphorylates EPO-stimulated EPO-R and participates in the down-regulation cascade of EPO-mediated signal transduction.
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PMID:Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling. 1452 37

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.
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PMID:Organization of functional domains in the docking protein p130Cas. 1548 52

PTP1B and TC-PTP are closely related protein tyrosine phosphatases, sharing 74% homology in their catalytic domain. However, their cellular localization, function, and regulation are found to be different. Their substrate specificity has implicated these enzymes in various signaling pathways, regulating metabolism, proliferation and cytokine signaling. For instance, PTP1B has been shown to regulate the activation of cytokine receptors through the dephosphorylation of specific members of the JAK family, namely JAK2 and TYK2, whereas TC-PTP is involved in the modulation of cytokine signaling via JAK1 and JAK3 molecules. Gene-targeting approaches will help us to unravel the physiological functions of these enzymes.
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PMID:Cytoplasmic protein tyrosine phosphatases, regulation and function: the roles of PTP1B and TC-PTP. 1578 May 98


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