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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals. In the past, the enzyme has been considered to be a protein Ser/Thr kinase although it was recently reported to contain Tyr(P) (Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993) EMBO J. 12, 803-808). A cDNA encoding rabbit skeletal muscle GSK-3 beta was cloned and expressed in Escherichia coli as an active protein kinase, with apparent M(r) 46,000, capable of phosphorylating several known GSK-3 substrates. Recombinant GSK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although the enzyme already contained Tyr(P) as judged by its recognition by anti-Tyr(P) antibodies. The net result of the autophosphorylation was a 3-5-fold reduction in enzyme activity. GSK-3 alpha, purified from rabbit muscle, also underwent autophosphorylation but only on Ser and Thr residues. In this case, the autophosphorylation stabilized the enzyme activity compared with the control lacking ATP/Mg2+. Of several phosphatases tested, the lambda-phage phosphatase was the most effective in dephosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues. The action of the lambda-phosphatase caused a reactivation of GSK-3 beta to approximately 80% of the starting activity. The protein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr residues leading to a reduction in enzyme activity. A truncated form of GSK-3 beta, apparent M(r) 40,000, had a significantly higher specific activity, was defective in autophosphorylation, and was not inactivated in the autophosphorylation reaction. We conclude that GSK-3 beta is a dual specificity protein kinase in the same sense as the mitogen-activated protein kinase/ERK family of enzymes. Phosphorylation at different residues differentially controls enzyme activity, Ser/Thr phosphorylation causing inactivation and Tyr phosphorylation resulting in increased activity.
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PMID:Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. 751 73

Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the beta PDGFR. The beta PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the beta PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P)-771 and -751, followed by Tyr(P)740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various rPTP1B displayed no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a beta PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type beta PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showign that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes.
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PMID:Identification of a putative Syp substrate, the PDGF beta receptor. 754 75

Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a phosphotyrosine-containing peptide derived from epidermal growth factor autophosphorylation site Tyr992 (EGFR988 998). This peptide photoinactivated PTP1 in a time- and concentration-dependent manner. Three lines of evidence indicate that the interaction between PTP1 and the photoaffinity label was specific: 1) photoinactivation was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr992 in molar excess. 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic constants similar to those of the non-Bpa-containing peptide under identical conditions. 3) After complete photoinactivation, the level of incorporation of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry of photolabeling. Radiolabeled peptide was used to identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and radioactive fragments were purified by high performance liquid chromatography (HPLC) and analyzed by Edman sequencing. In two parallel experiments which were analyzed using different HPLC columns, a site in the alpha2 region of PTP1, most likely Ile23, was labeled by the Tyr992-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of substrate recognition must exist for PTP1 catalysis.
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PMID:Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1. 862 92

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.
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PMID:Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. 956 38

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.
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PMID:Protein tyrosine phosphatase PTP1B suppresses p210 bcr-abl-induced transformation of rat-1 fibroblasts and promotes differentiation of K562 cells. 982 59

Monocyte adhesion resulted in rapid tyrosine phosphorylation and subsequent cytokine mRNA induction. The objective of this study was to determine the role of specific tyrosine phosphorylation events, particularly those involving members of the MAP kinase family, in regulating adhesion-induced cytokine expression. Using nuclear run-on analyses, we demonstrated that on adhesion, monocytes rapidly transcriptionally activated numerous cytokine mRNAs, coincident with the activation of the transcription factors NF-KB and AP-1. Both an inhibitor of tyrosine phosphorylation, genistein, and the cytoplasmic tyrosine phosphatase PTP1B, were unable to prevent adhesion-mediated transcriptional activation. However, both blocked adhesion-induced ERK and JNK but not p38 kinase activation and at the same time decreased the stability of interleukin-1beta (IL-1beta) and IL-8 transcripts. In addition, whereas adhesive events occurred in the presence of genistein and PTP1B, monocyte spreading was markedly inhibited. Our results suggest that the majority of protein phosphorylation events are associated with adhesion-induced cytokine expression through transcript stabilization and cytoskeletal organization. A minority of protein phosphorylation events, not sensitive to genistein or PTP1B exposure, may be instrumental in regulating transcription. Thus the spectrum of protein tyrosine kinases required for transcription appear distinct from those involved in maintaining the stability of some cytokine mRNAs and the integrity of the cytoskeleton to which mRNA destined for translation must be associated.
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PMID:Differential role of tyrosine phosphorylation in adhesion-induced transcription, mRNA stability, and cytoskeletal organization in human monocytes. 1067 May 83

In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin alpha subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1.
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PMID:The association of CRKII with C3G can be regulated by integrins and defines a novel means to regulate the mitogen-activated protein kinases. 1077 17

Vascular smooth muscle cell (VSMC) migration and growth are positively regulated by protein tyrosine phosphorylation. Therefore, a dephosphorylation process controlled by protein tyrosine phosphatases (PTPs) must also be critical. The present study identified six cytoplasmic PTPs expressed in VSMCs: low M(r) protein tyrosine phosphatase (LMW-PTP), SHP-2, PTP36, PTP2, PTP1B, and FAP1. We further examined the functions of LMW-PTP in VSMCs using the adenovirus-mediated gene transfer of recombinant LMW-PTP. PDGF-induced activation of p38, but not of ERK MAP kinase, was blocked by LMW-PTP. LMW-PTP as well as the p38 inhibitor SB203580 inhibited DNA synthesis and cell migration upon PDGF stimulation. LMW-PTP dephosphorylated activated PDGF receptors in NIH3T3 cells, but not in VSMCs. Thus, LMW-PTP negatively regulates PDGF functions by inhibiting the p38 pathway in VSMCs although its substrate is unclear. These findings strongly demonstrate that PTPs are important as negative regulators for VSMC growth and migration, processes that are closely related to the progression of atherosclerosis.
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PMID:Low M(r) protein tyrosine phosphatase inhibits growth and migration of vascular smooth muscle cells induced by platelet-derived growth factor. 1171 18

Protein tyrosine phosphatase (PTP) in-gel assays were used to explore association of PTPs with the platelet-derived growth factor beta-receptor (PDGFbetaR). Five PTP activity bands of approximately 120, approximately 70, approximately 60, approximately 53, and approximately 45 kDa could be detected in PDGFbetaR immunoprecipitates and were identified by immunodepletion experiments as PTP-PEST, SHP-2, an active fragment of SHP-2, PTP-1B, and T-cell PTP, respectively. The PTP pattern that was obtained was similar in PDGFbetaR immunoprecipitates from HEK 293 cells overexpressing the human PDGFbetaR and from murine fibroblasts. Association of PTP-1B with the PDGFbetaR was stabilized by pretreatment of the cells with hydrogen peroxide. The epidermal growth factor receptor (EGFR) immunoprecipitated from fibroblasts, and c-Kit isolated from CHRF myeloid cells, were associated with partially overlapping but quantitatively different patterns of PTPs. PTP-PEST was the predominant PTP in EGFR immunoprecipitates, and SHP-1 appeared in c-Kit immunoprecipitates. We propose that the differential association of PTPs with different RTKs is related to their respective contributions to regulation of RTK signaling.
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PMID:Identification of protein tyrosine phosphatases associating with the PDGF receptor. 1261 64

The intracellular production of reactive oxygen species (ROS) has a fundamental importance in both cell proliferation and apoptosis induction. Moreover, many experimental and epidemiological evidence indicate that ROS contribute to the initiation and promotion of carcinogenesis, and that drugs or treatments aimed to reduce the tissue content of ROS can be chemopreventive and curative against cancer. Recently, important observations on the role of ROS as physiological regulators of intracellular signaling cascades activated by growth factors through their tyrosine-kinase receptors have shed new light on the possible mechanisms that can sustain the promoting activity of ROS. The downstream effect of ROS production is the reversible oxidation of proteins. Redox sensitive proteins include protein tyrosine phosphatases (PTPs) as the active-site cysteine is the target of specific oxidation, and this modification can be reversed by intracellular reducing agents. The reversible oxidation of PTPs family member was demonstrated firstly for PTP1B during EGF signaling and then for LMW-PTP and SHP-2 during PDGF stimulation. The inhibition exerted by ROS on tyrosine-phosphatases helps the propagation of RTK signals mediated by protein tyrosine phosphorylation, generally associated with the proliferative stimulus. Our new data are consistent with a model in which ROS take a role in integrin signaling, and in which synergistic activation of Rac-1 by growth factors and adhesion molecules translates in a critical increase of intracellular oxidants up to a threshold level where inhibition of the tyrosine phosphatase LMW-PTP takes place. In seeking for potential molecular mechanisms for oxidative signaling by integrins, we found that transient oxidation/inactivation of LMW-PTP, a known negative regulator of RTK signaling, occurred during fibroblast adhesion to matrix, with a kinetic which paralleled the generation of ROS. Moreover, overexpression of LMW-PTP in NIH-3T3 fibroblasts delayed cell attachment to the substrate. Finally, constitutively high levels of intracellular ROS, as are observed in cells expressing active Rac, would attenuate anchorage dependence for growth, by substituting for integrin signaling in non adherent cells.
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PMID:Reactive oxygen species as mediators of cell adhesion. 1283 35


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