Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51812 (mitogen-activated protein)
 10,636 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The p53 tumor suppressor protein is tightly regulated in the cell and is phosphorylated at multiple sites by several different protein kinases. We have investigated the phosphorylation of p53 by mitogen-activated protein (MAP) kinase, a protein kinase that plays a central role in mediating many mitogenic and differentiation signals. Recombinant wild-type mouse p53 was phosphorylated in vitro by activated recombinant p42-MAP kinase but not by inactive MAP kinase or by the activating protein, MAP kinase kinase. Phosphorylation of p53 by MAP kinase occurred at two N-terminal sites, threonine residues 73 and 83. Tryptic phosphopeptides of recombinant p53 phosphorylated in vitro by MAP kinase comigrated on two-dimensional maps with p53 from SV3T3 cells labeled in vivo with [32P]orthophosphate, suggesting that MAP kinase targets a site in p53 that is phosphorylated in the cell. Following serum stimulation of quiescent C57MG cells, two p53 kinases, which were resolved by chromatography on Mono Q, were stimulated 15-20-fold within 5 min. Each of these kinase activities co-eluted with myelin basic protein kinase activity and could be inactivated following treatment with protein phosphatase 2A, a serine/threonine phosphatase, or leukocyte antigen receptor, a protein tyrosine phosphatase, suggesting that these activities were members of the MAP kinase family. The two kinase activities from the lysates targeted the same phosphorylation sites on p53 as the purified recombinant MAP kinase. These protein kinase activities were also stimulated following exposure of the cells to ultraviolet radiation, but with slightly delayed kinetics. Phorbol ester treatment of SV3T3 cells led to increased phosphorylation of the peptide containing the residues targeted by MAP kinase. The data suggest that p53 may be phosphorylated by MAP kinase physiologically and that this interaction may be involved in the cell's response to UV exposure, growth factor stimulation, or transformation by oncogenes.
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PMID:Phosphorylation of the tumor suppressor protein p53 by mitogen-activated protein kinases. 751 Jul 6

The precise role of the protein tyrosine phosphatase Syp in insulin signaling is not well understood. We previously reported that expression of catalytically inactive Syp phosphatase blocked stimulation of mitogen-activated protein (MAP) kinase by insulin. In this study, we investigated the effect of dominant negative Syp on the intermediates in MAP kinase pathway. The expression of dominant negative Syp blocked the activation of MEK and raf-1 kinase in response to insulin and had no detectable effect on insulin-induced activation of p21ras. These data suggest that the target of the Syp phosphatase may reside in proteins immediately downstream of p21ras.
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PMID:Expression of a catalytically inert Syp blocks activation of MAP kinase pathway downstream of p21ras. 767 89

Sublethal concentrations of reactive oxygen intermediates including H2O2 can alter human T cell function and inhibit proliferative responses but relatively little is known about the effects of low levels of oxidant stress on signaling pathways. In the present study, we investigated whether the exposure of Jurkat T cells to micromolar concentrations of H2O2 might influence the activity of certain serine/threonine kinases and protein phosphatases important for T cell signaling as well as initiation of nuclear events. Jurkat cells treated with 100-200 microM H2O2 exhibited rapid increases in cytosolic protein kinase C (PKC) activity without detectable translocation of PKC to the membrane/particulate compartment. The stimulation of PKC activity by H2O2 was associated with an increase in the activation of kinases phosphorylating myelin basic protein (MBP), a substrate for mitogen-activated protein (MAP) kinase and RRLSSLRA (S6 peptide; a substrate for the approximately 90-kDa ribosomal S6 kinases). Optimal activation of MAP kinase in cells treated with H2O2 was preceded by increases in protein tyrosine phosphorylations and occurred at sublethal concentrations of H2O2 which did not markedly deplete intracellular ATP. Pretreatment of cells with the PKC inhibitors sangivamycin and H7 suppressed but did not block the stimulation of MAP kinase activity in response to H2O2 or phytohemagglutinin. The activities of both protein tyrosine phosphatase (PTP) and protein phosphatase 2A (PP2A) were reduced after H2O2 treatment of intact cells. Furthermore, kinetic studies showed that H2O2 was capable of suppressing the activities of PTP and PP2A before inducing optimal increases in MAP kinase activity. These results demonstrate that the exposure of T cells to sublethal levels of oxidant stress acutely stimulates the MAP kinase cascade and suggest that this activation may involve PKC-dependent and -independent pathways as well as inhibition of certain protein phosphatases.
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PMID:Sublethal levels of oxidant stress stimulate multiple serine/threonine kinases and suppress protein phosphatases in Jurkat T cells. 777 89

An expression and purification method was developed to obtain the recombinant human dual-specific protein tyrosine phosphatase (PTPase) VHR in quantities suitable for both kinetic studies and crystallization. Physical characterization of the homogeneous recombinant protein verified the mass to be 20,500 +/- 100 by matrix-assisted laser desorption mass spectrometry, confirmed the anticipated NH2-terminal amino acid sequence and demonstrated that the protein exists as a monomer. Conditions were developed to obtain crystals which were suitable for x-ray structure determination. Using synthetic diphosphorylated peptides corresponding to MAP177-189 (mitogen-activated protein) kinase (DHTG-FLpTEpYVATR), an assay was devised which permitted the determination of the rate constants for dephosphorylation of the diphosphorylated peptide on threonine and tyrosine residues. The diphosphorylated peptides are preferred over the singly phosphorylated on tyrosine by 3-8-fold. The apparent second-order rate constant kcat/Km for dephosphorylation of phosphotyrosine on DHTGFLpTEpYVATR was 32,000 M-1 S-1 while dephosphorylation of phosphothreonine was 14 M-1 S-1 (pH 6). The reaction of DHTGFLpTEpYVATR with VHR is ordered, with rapid dephosphorylation on tyrosine occurring first followed by slow dephosphorylation on threonine. Similar results were obtained with F(NLe)(N-Le)pTPpYVVTR, a peptide corresponding to a MAP kinase-like protein (JNK1(180-189)) which is involved in the stress response signaling pathway.
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PMID:The purification and characterization of a human dual-specific protein tyrosine phosphatase. 787 21

In addition to their role in bacterial killing, reactive oxygen intermediates (ROI) produced by the NADPH oxidase may participate in the regulation of intracellular pathways. We have recently demonstrated that ROI produced by the oxidase regulate tyrosine phosphorylation in neutrophils, possibly by alterations in the cellular redox state. The purpose of the present study was to characterize the identities of certain of the redox-sensitive tyrosine-phosphorylated substrates and the significance of the increased phosphorylation. As a prominent 42-44-kDa phosphorylated band was noted in oxidant-treated cells, we investigated the possible phosphorylation and activation of mitogen-activated protein (MAP) kinase under these conditions. Immunoprecipitation of MAP kinase followed by immunoblotting with anti-phosphotyrosine antibodies indicated that a 42-44-kDa polypeptide was tyrosine-phosphorylated in response to treatment of cells, either with the oxidizing agent diamide or with H2O2 in cells where catalase was inhibited. Using an in vitro renaturation assay with myelin basic protein as the substrate, oxidant-induced stimulation of kinase activity of a 42-44-kDa band was observed in both whole cell extracts and in MAP kinase immunoprecipitates. The mechanism of redox-sensitive activation of MAP kinase was examined. First, exposure of cells to oxidants caused a significant increase in the activity of MEK (the putative activator of MAP kinase), as determined by an in vitro kinase assay using recombinant catalytically inactive glutathione S-transferase-MAP kinase as the substrate. Additionally, oxidant treatment of cells resulted in inhibition of the activity of CD45, a protein tyrosine phosphatase known to dephosphorylate and inactivate MAP kinase. We conclude that oxidant treatment of neutrophils can activate MAP kinase by stimulating its tyrosine and (presumably) threonine phosphorylation via MEK activation, a response that may be potentiated by inhibition of MAP kinase dephosphorylation by phosphatases such as CD45.
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PMID:Activation of the mitogen-activated protein kinase signaling pathway in neutrophils. Role of oxidants. 798 67

The mitogen-activated protein (MAP) kinases are serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation by the dual specificity protein kinase MEK (MAP kinase/ERK kinase). The present report describes the purification to near homogeneity and characterization of a protein tyrosine phosphatase from Xenopus laevis eggs that dephosphorylates MAP kinase phosphorylated by MEK. Bacterially expressed Xenopus MAP kinase phosphorylated by purified Xenopus MEK was used as substrate throughout the purification. The purification procedure included anion-exchange, cation-exchange, gel filtration, heparin-Sepharose, and chromatography on a column of thiophosphorylated MAP kinase-Sepharose, resulting in a > 3000-fold purification. Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein of 47 kDa correlated with activity. The phosphatase showed absolute specificity toward phosphotyrosine and no activity toward phosphothreonyl-phosphoseryl residues of MAP kinase. The pH optimum of the enzyme was 7.0 with a Km of 9.0 microM for phosphorylated MAP kinase. The phosphatase was inhibited by ammonium molybdate (IC50, 2 microM), vanadate (IC50, 250 microM), millimolar concentrations of MnCl2, ZnCl2 and p-nitrophenylphosphate but not by okadaic acid or microcystin. This tyrosine phosphatase may be involved in deactivating MAP kinase in vivo.
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PMID:Purification and characterization of a mitogen-activated protein kinase tyrosine phosphatase from Xenopus eggs. 822 71

The dual specificity protein-tyrosine phosphatase rVH6 belongs to a subfamily of enzymes that have in vivo and in vitro catalytic activity against mitogen-activated protein kinases. A method was developed for the expression and efficient purification of recombinant rVH6 in quantities sufficient for physical and kinetic characterization of the enzyme. Matrix-assisted laser desorption mass spectrometry verified the mass of purified rVH6 to be 43,500 +/- 150, and NH2-terminal sequence analysis confirmed the predicted amino acid sequence. Kinetic characterization of full-length rVH6 identified the critical ionizations involved in the kcat/Km parameter (apparent pKa values 5.1 and 6.6) and revealed a pH-independent kcat value of 0.014 s-1. In an attempt to define the essential catalytic core of this enzyme, amino acids 134-381 of rVH6 were expressed, purified, and characterized enzymatically. Kinetic analysis revealed that the truncated enzyme exhibited a turnover value similar to that of the full-length enzyme (kcat = 0.017 s-1), with p-nitrophenyl phosphate as substrate. Secondary structure prediction and molecular modeling of rVH6 based on the x-ray structure of the dual specificity protein tyrosine phosphatase, VHR, further supported the assignment of residues 134-381 to the core catalytic domain of rVH6. These results demonstrate that the NH2 terminus of rVH6 (residues 1-133) is not required for full enzyme activity and comprises a separate domain that may play a distinct physiological function.
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PMID:Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6. 896 12

Angiotensin II type 2 (AT2) receptors are involved in the inhibition of cell proliferation as well as in apoptosis and neuronal differentiation, through intracellular signalling pathways that remain poorly defined. The present study examines the effect of AT2-receptor stimulation on growth-factor-induced pathways leading to the activation of mitogen-activated protein (MAP) kinases. In N1E-115 neuroblastoma cells, AT2 receptors inhibit the activity of MAP kinases induced by serum as well as by epidermal growth factor. The inhibitory effect of angiotensin II (Ang II) is rapid and transient, and affects both ERK1 and ERK2 (extracellular signal-related protein kinase) isoforms of the enzyme. AT2-mediated MAP kinase inactivation is not sensitive to pertussis toxin or okadaic acid, but involves a vanadate-sensitive protein tyrosine phosphatase (PTP). Expression of MAP kinase phosphatase-1 (MKP-1) is not significantly modified upon AT2-receptor activation, and insensitivity to actinomycin D also rules out transcriptional induction of other MKPs as a possible mechanism for AT2-mediated inactivation of MAP kinases. In addition, we report here that both in N1E-115 cells and in Chinese hamster ovary cells expressing recombinant human AT2 receptors, Ang II rapidly stimulates the catalytic activity of SHP-1, a soluble PTP that has been implicated in termination of signalling by cytokine and growth-factor receptors. These findings thus demonstrate functional negative cross-talk between heptahelical AT2 receptors and receptor tyrosine kinases, and suggest that SHP-1 tyrosine phosphatase is an early transducer of the AT2 receptor signalling pathway.
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PMID:Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase. 923 Jan 27

There is growing evidence for the role of protein tyrosine phosphatases in controlling such fundamental cellular processes as growth and differentiation. Pervanadate is a potent inhibitor of protein tyrosine phosphatase which has been observed here to induce proliferation in C3H10T1/2 mouse fibroblasts. Pervanadate also translocated/activated p42/44 mitogen-activated protein (MAP) kinase to the cell nucleus. An almost similar pattern of nuclear p42/44 MAP kinase stimulation is seen with TPA. On the other hand, TPA treatment results in a rapid activation of cytosolic MAP kinase which declines with time. Thus pervanadate appears as a very useful tool for studying tyrosine phosphorylation.
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PMID:Pervanadate elicits proliferation and mediates activation of mitogen-activated protein (MAP) kinase in the nucleus. 927 39

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.
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PMID:Early signaling events triggered by peroxovanadium [bpV(phen)] are insulin receptor kinase (IRK)-dependent: specificity of inhibition of IRK-associated protein tyrosine phosphatase(s) by bpV(phen). 941 95


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