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:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of mitogen-activated protein (MAP) kinase represents an important mechanism in hormonal regulation. To clarify the role of MAP kinase activation in insulin action, we compared the activation of the enzyme in Rat-1 cells transfected with wild-type (Hirc) and mutant insulin receptors in which the 2 carboxyl-terminal tyrosines were substituted with phenylalanine (Y/F2). Expression of the Y/F2 mutant receptor enhanced the responsiveness of MAP kinase to insulin. Moreover, the insulin responsiveness of the activator of this enzyme, MAP kinase kinase, was also increased in these cells. To explore the early signaling events that might account for this increase in responsiveness, we evaluated the tyrosine phosphorylation of the insulin receptor substrate, IRS-1, and its subsequent association with phosphatidylinositol (PI)-3 kinase. In both cell types, insulin led to a dose-dependent increase in the association of tyrosine phosphorylated IRS-1 with the SH2 domain of the p85 regulatory subunit of PI-3 kinase, and also increased the amount of PI kinase activity detected in anti-IRS-1 immunoprecipitates. The effect of insulin was significantly greater in Y/F2 cells, as determined in both assays. In previous studies, cells bearing this receptor mutant exhibited an identical metabolic response but enhanced mitogenic response to insulin when compared with wild-type receptor. These data provide further evidence for divergence of the mitogenic and metabolic signaling pathways at or near the insulin receptor.
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PMID:Mutation of the two carboxyl-terminal tyrosines in the insulin receptor results in enhanced activation of mitogen-activated protein kinase. 814 49

GRB-2 is a small SH2- and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p21ras during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-1. Coexpression of IRS-1 or IRS-1F-895 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21ras and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-895, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21ras-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.
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PMID:Role of IRS-1-GRB-2 complexes in insulin signaling. 819 3

The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except that it could tolerate replacement of the arginine by a lysine and the phosphorylation-site serine by a threonine residue. Partial cDNAs encoding MAPKAP kinase-2 were isolated from rabbit and human skeletal muscle and human teratocarcinoma libraries, and Northern-blotting experiments revealed a single 3.3 kb mRNA transcript present at similar levels in six human tissues examined. The catalytic domain was most similar (35-40% identity) to calmodulin-dependent protein kinases II and IV, phosphorylase kinase, putative serine kinase H1 and the C-terminal domain of MAPKAP kinase-1, which form one branch of the protein kinase phylogenetic tree. The sequence N-terminal to the catalytic domain is proline-rich and contains two putative SH3-binding sites. The threonine residue phosphorylated by MAP kinase lies immediately C-terminal to the catalytic domain and is followed by a nuclear localization signal, Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys, near the C-terminus.
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PMID:The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. 828 84

Activation of the microbicidal response of phagocytes requires cytosolic ATP and is associated with extensive protein phosphorylation, suggesting the involvement of protein kinases in the signal transduction cascade. An in vitro renaturation assay was used to identify the protein kinase(s) activated by chemoattractants in human blood neutrophils. Four distinct kinases were activated by the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine with molecular masses of 72, 65, 49, and 41 kDa (designated PK72, PK65, PK49, and PK41, respectively). PK72 and PK65 were activated very rapidly (5-15 s), yet transiently. By comparison, PK49 and PK41 responded in a slower, more sustained manner. Treatment of extracts of activated cells with alkaline phosphatase reverted the stimulation of the kinases, suggesting that phosphorylation is the post-translational modification that underlies activation of the kinases. Stimulation of PK72 and PK65 by chemoattractant was independent of calcium and protein kinase C. In contrast, elevation of cytosolic free calcium levels was sufficient and appeared to be necessary for full activation of PK49 and PK41. While phorbol esters can mimic the effects of formyl-methionyl-leucyl-phenylalanine on PK49 and PK41, inhibition of protein kinase C by staurosporine did not prevent the receptor-mediated activation of these kinases. PK41 most likely corresponds to the Erk-1 isoform of mitogen-activated protein (MAP) kinase. Accordingly, PK41 effectively phosphorylated myelin basic protein, known to be a good substrate for Erk-1. The electrophoretic mobility of PK49 is similar to that of MAP kinase-kinase (MAP/Erk kinase). However, immunoprecipitation experiments indicated that PK49 is not MAP/Erk kinase. The identity of this and other kinases remains to be defined, but possible candidates are discussed. In addition to autophosphorylating, PK72, PK65, and PK41 were shown to effectively phosphorylate exogenous substrates. These kinases may therefore play a role in signal transduction during stimulation by chemoattractants.
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PMID:Receptor-mediated activation of multiple serine/threonine kinases in human leukocytes. 837 83

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.
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PMID:A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C. 838 19

Mutant epidermal growth factor (EGF) receptors in which the five known tyrosine autophosphorylation sites (tyrosines 992, 1068, 1086, 1148, and 1173) were replaced with phenylalanine residues were expressed in NIH-3T3 cells (5F-EGFR) and transmembrane signaling parameters compared with cells expressing wild-type EGF receptor (WT-EGFR). Mutant and wild-type clones were chosen expressing similar numbers of receptors and Scatchard analysis of 125I-EGF binding showed high and low affinity binding of equal affinities for both receptor types. EGF stimulated tyrosine phosphorylation of proteins to a much lesser degree in cells expressing 5F-EGFR relative to cells expressing WT-EGFR. Tyrosine phosphorylation of the 5F-EGFR was 2-4% of WT-EGFR. Surprisingly, cells expressing WT-EGFR or 5F-EGFR showed little difference in dose response of EGF-stimulated [3H]thymidine incorporation or EGF stimulation of mitogen-activated protein kinase activity. However, EGF did not induce anchorage-independent growth of cells expressing 5F-EGFR to the same extent as it did for cells expressing WT-EGFR. EGF treatment of 5F-EGFR cells failed to elicit an increase in phosphatidylinositol 3-kinase activity or to stimulate hydrolysis of phosphoinositides or tyrosine phosphorylation of phospholipase C-gamma 1. These data suggest that a significant proportion of EGF receptor signaling can occur through receptors with altered capacity to interact with src homology 2 domain-containing proteins.
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PMID:Transmembrane signaling by epidermal growth factor receptors lacking autophosphorylation sites. 838 84

We have studied the functions of the juxtamembrane domain (941-989) of the human insulin receptor by site-directed mutagenesis. Tyrosine phosphorylation of pp185 was impaired in Chinese hamster ovary cells expressing the receptors with the alteration of Tyr960, but not of Tyr953 or Tyr972, to Phe (CHO-Y960F cells) as compared with cells expressing the normal receptors. In CHO-Y960F cells, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the activation of phosphatidylinositol 3-kinase in the anti-phosphotyrosine and anti-IRS-1 immunoprecipitates, the activation of mitogen-activated protein (MAP) kinase, and biological actions were also impaired. In addition, although the deletion of residues 954-965 severely impaired insulin internalization, the deletion of NPXY (957-960), the internalization signal of the low density lipoprotein receptor, did not affect internalization. Moreover, neither the deletions around Tyr953 nor the alterations of the tyrosines (953, 960, or 972) significantly reduced internalization. These data suggest that: 1) Tyr960 is important for the recognition of pp185/IRS-1, the association of phosphatidylinositol 3-kinase with pp185/IRS-1, and the activation of MAP kinase; 2) MAP kinase may lie downstream of pp185/IRS-1 in insulin's signal transduction; and 3) the juxtamembrane domain, but not NPXY or individual tyrosines, is important for insulin internalization.
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PMID:Site-directed mutagenesis of the juxtamembrane domain of the human insulin receptor. 839 70

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.
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PMID:Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1. 844 9

Signal transduction from the granulocyte colony-stimulating factor receptor (G-CSF-R) occurs via multiple pathways, one of which involves activation of p21Ras and mitogen-activated protein kinase. The SH2 domain-containing proteins Shc and GRB2 have been implicated in this latter signaling route. We studied the role of these proteins in signal transduction from wild type (WT) G-CSF-R, C-terminal deletion mutants, and tyrosine-to-phenylalanine substitution mutants in transfectants of the mouse pro-B cell line, BAF3. G-CSF stimulation of BAF3 cells expressing WT G-CSF-R induced tyrosine phosphorylation of Shc. Anti-Shc antibodies co-immunoprecipitated tyrosine-phosphorylated 145-kD proteins (p145), whereas GRB2 immunoprecipitates contained phosphorylated Shc, Syp, and proteins of 145 and 90 kD (p90). Neither of these complexes were detected after activation of a C-terminal deletion mutant of G-CSF-R that lacked all four conserved cytoplasmic tyrosine residues. G-CSF induced formation of Syp/GRB2 complexes in all the tyrosine-substitution mutants, suggesting that this association did not depend on the presence of single specific tyrosine residues in G-CSF-R. In contrast, tyrosine 764 of G-CSF-R appeared to be exclusively required for tyrosine phosphorylation of Shc and its association with p145 and GRB2. In addition, tyrosine 764 also specifically mediated binding of GRB2 to p90 without the involvement of Shc. These findings indicate that tyrosine 764 of G-CSF-R has a prominent role in G-CSF signal transduction.
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PMID:Specific involvement of tyrosine 764 of human granulocyte colony-stimulating factor receptor in signal transduction mediated by p145/Shc/GRB2 or p90/GRB2 complexes. 854 34

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.
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PMID:Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression. 862 63


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