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)

We have studied the function of a mutant human insulin receptor in which two COOH-terminal autophosphorylation sites (Tyr-1316 and -1322) were replaced by phenylalanine (F/Y COOH-terminal 2 tyrosines (CT2)). In addition, we have also constructed a mutant receptor in which Lys-1018 in the ATP-binding site was changed to arginine (R/K 1018). Both the wild type insulin receptor (HIR) and the mutant receptors were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation of solubilized and partially purified F/Y CT2 was decreased by approximately 30% compared with the HIR. Tyrosine kinase activities of F/Y CT2 and HIR toward exogenous substrates were almost equal. When CHO cells transfected with F/Y CT2 (CHO-F/Y CT2) were stimulated with insulin, autophosphorylation of the beta-subunit of the insulin receptor and the phosphorylation of an endogenous substrate (pp185) in the intact cell were normal compared with cells expressing HIR (CHO-HIR). CHO-F/Y CT2 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. However, the dose-response curve of insulin-stimulated thymidine incorporation in CHO-F/Y CT2 was shifted to the left (approximately 5-7-fold) compared with that in CHO-HIR. There was no significant difference in insulin-like growth factor 1-stimulated thymidine incorporation between CHO-F/Y CT2 and CHO-HIR. Furthermore, the dose-response curve of insulin-stimulated kinase activity toward myelin basic protein in CHO-F/Y CT2 was also shifted to the left (approximately 5-fold) compared with that in CHO-HIR. Kinase assays in myelin basic protein-containing gels revealed that both species of MAP kinases (M(r) 44,000, 42,000) were more sensitive to activation by insulin in CHO-F/Y CT2 than in CHO-HIR. This observation was confirmed in immune complex kinase assays toward microtubule-associated protein 2 (MAP2) using specific antibodies against mitogen-activated protein (MAP) kinase. R/K 1018 mutant insulin receptors showed an absence of insulin-stimulated kinase activity and CHO cells transfected with R/K 1018 (CHO-R/K 1018) failed to enhance 2-deoxyglucose uptake or thymidine incorporation in response to insulin. In addition, R/K 1018 kinase-defective insulin receptors were unable to mediate insulin-stimulated MAP kinase activation. These data suggest that: 1) tyrosine kinase activity of the insulin receptor is required for activation of insulin-stimulated MAP kinases and 2) phosphorylation of COOH-terminal tyrosine residues may play an inhibitory role in mitogenic signaling through regulation of MAP kinases.
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PMID:Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine. 161 80

Activation of the mitogen-activated protein (MAP) kinase pathway is believed to play a critical role in normal and pathophysiological proliferation of mesangial cells. Recent studies have shown that MAP kinase activation by growth factors in other cell types involves activation of the low-molecular-weight G protein Ras and the protooncogene serine kinase c-Raf-1. In this study, the role of this pathway in rat renal mesangial cells was assessed. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), as well as phorbol esters (PMA) rapidly activated MAP kinase three- to fourfold in these cells. PDGF and EGF, but not PMA, were able to activate c-Raf-1 and Ras activity. Stimulation of mesangial cells with the inflammatory mediator prostaglandin E2 (PGE2) or elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by treatment with forskolin markedly blunted activation of MAP kinase induced by PDGF and EGF, but not by PMA. Consistent with this observation, PGE2 abolished growth factor-induced activation of c-Raf-1. However, Ras activation induced by growth factors was not affected by PGE2 and forskolin. These results suggest that MAP kinase activation can occur by at least two separate pathways in mesangial cells. Tyrosine kinase receptors activate MAP kinase through activation of Ras and Raf. This pathway can be blocked by PGE2 and elevation of cAMP, presumably by interfering with the ability of Ras to activate Raf. In addition, activation of protein kinase C by phorbol esters can activate MAP kinase in a Ras/Raf-independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of MAP kinase by prostaglandin E2 and forskolin in rat renal mesangial cells. 748 69

In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.
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PMID:Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. 752 42

Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti-integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin-mediated focal accumulation of other cytoskeletal molecules including F-actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.
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PMID:Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. 759 97

Activation of the mitogen-activated protein kinase (MAPK) pathway is believed to play a critical role in normal and pathophysiologic proliferation of mesangial cells. Recent studies have shown that MAP kinase activation by growth factors in other cell types involves activation of the low molecular weight G-protein ras and the protooncogene serine kinase c-raf-1. In this study the role of this pathway in rat renal mesangial cells was assessed. 20ng/ml of platelet-derived growth factor (PDGF), 10(-8) mol/L epidermal growth factor (EGF) as well as phorbol ester (10(-6) mol/L PMA) rapidly activated MAP kinase by 3-4 fold in these cells. PDGF and EGF, but not PMA were able to activate c-raf-1 and ras activity. Stimulation with inflammatory mediator PGE2 (50 mumol/L) or elevation of Intracellular cAMP by treatment of cells with forskolin (25 mumol/L) markedly blunted activation of MAP kinase induced by PDGF and EGF, but not PMA. Consistent with this observation, PGE2 abolished growth factor induced activation of c-raf-1. However, ras activation induced by growth factor was not affected by PGE2 and forskolin. These results suggest that MAP kinase activation can occur by at least two separate pathways in mesangial cells. Tyrosine kinase receptors activate MAP kinase through activation of ras and raf. This pathway can be blocked by PGE2 and elevation of cAMP, presumably by interfering with the ability of ras to activate raf. In addition, activation of protein kinase C by phorbol esters can activate MAP kinase in a ras/raf-independent manner. This pathway is not sensitive to inhibition by PGE2 or cAMP. It is likely that activation of each of these pathways, both resulting in a stimulated MAP kinase, will have different physiologic consequences in mediating mesangial cells growth.
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PMID:[Inhibition of growth factor stimulation of mitogen-activated protein kinase by prostaglandin E2 in rat renal mesangial cells]. 778 49

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.
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PMID:Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. 841 57

Plasmalopsychosine, a characteristic fatty aldehyde conjugate of beta-galactosylsphingosine (psychosine) found in brain white matter, enhances p140trk (Trk A) phosphorylation and mitogen-activated protein kinase (MAPK) activity and as a consequence induces neurite outgrowth in PC12 cells. The effect of plasmalopsychosine on neurite outgrowth and its prolonged activation of MAPK was similar to that of nerve growth factor (NGF), and the effect was specific to neuronal cells. Plasmalopsychosine was not capable of competing with cold chase-stable, high affinity binding of NGF to Trk A, indicating that plasmalopsychosine and NGF differ in terms of Trk A-activating mechanism. Tyrosine kinase inhibitors K-252a and staurosporine, known to inhibit the neurotrophic effect of NGF, also inhibited these effects of plasmalopsychosine, suggesting that plasmalopsychosine and NGF share a common signaling cascade. Plasmalopsychosine prevents apoptosis of PC12 cells caused by serum deprivation, indicating that it has "neurotrophic factor-like" activity. Taken together, these findings indicate that plasmalopsychosine may play an important role in development and maintenance of the vertebrate nervous system.
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PMID:Plasmalopsychosine of human brain mimics the effect of nerve growth factor by activating its receptor kinase and mitogen-activated protein kinase in PC12 cells. Induction of neurite outgrowth and prevention of apoptosis. 855 9

Breast cancers frequently over-express a number of growth factor receptors. In addition, elevated src family kinase activity is present in a percentage of these neoplasms and has been implicated in signal transduction in these cells. Therefore, inhibiting tyrosine kinase activity is a potential approach for treating these tumors. Utilizing the SKBR3 and MCF-7 breast cancer cell lines, we evaluated the effects of broadly targeting growth factor receptor and cytoplasmic tyrosine kinases with tyrosine kinase inhibitors (herbimycin A and genistein) to inhibit proliferation. We also evaluated these inhibitor's effects on proteins that regulate ras function, which is a convergence point for signaling through both src family kinases and a number of growth factor receptors with tyrosine kinase activity (e.g., epidermal growth factor and erbB-2 receptors). We specifically evaluated whether these compounds affected 2 recently discovered proteins involved in controlling ras function: Shc, which is tyrosine-phosphorylated by src and activated growth factor receptors, and Grb-2, which mediates signal transduction from activated growth factor receptors through ras. We evaluated their effects on tyrosine phosphorylation of Shc, binding of Grb-2 to Shc and MAP kinase activity. Both cell lines were inhibited in a dose-dependent manner by each compound. This was accompanied by decreased Shc tyrosine phosphorylation, Shc's association with Grb-2 and MAP kinase activity. Thus, tyrosine kinase inhibitors can inhibit proliferation of breast cancer cells, accompanied by inhibition of signal transduction steps potentially mediated through ras. Tyrosine kinase inhibitors might, therefore, be useful for the treatment of breast cancer.
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PMID:Effects of tyrosine kinase inhibitors on the proliferation of human breast cancer cell lines and proteins important in the ras signaling pathway. 856 15

Angiotensin II (Ang II) is a potent regulator of proximal tubule functions, including transport, metabolism, and cell proliferation. The opossum kidney (OK) cell line is a useful model of renal proximal tubule. Mitogen-activated protein (MAP) kinases are rapidly phosphorylated and activated in response to various agonists. We investigated Ang II effects on serine/threonine kinase cascades in OK cells. The major findings of the present study are that Ang II stimulated MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase activities, and that it increased phosphorylation of Raf-1 kinase and p42 MAP kinase in OK cells. These stimulations of kinases were dose-dependent (from 10(-6) to 10(-11) M). The time course of activation was sequential; the peak stimulation was reached at 5 to 10 minutes for Raf-1 kinase, MAPKK and MAPK, and at 20 minutes for S6 kinase. The activation of MAPK was inhibited by approximately 70% with prolonged 24-hour PMA pretreatment or in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein and herbimycin) did not inhibit AngII-induced MAPK activity. This activation of MAPK was also inhibited via AT1 receptor antagonist, Dup753 and pertussis toxin. This evidence suggests that the activation of serine/threonine cascades by Ang II is largely dependent on PMA-sensitive PKC, and is not dependent on tyrosine kinase and pertussis toxin.
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PMID:Sequential activation of MAP kinase cascade by angiotensin II in opossum kidney cells. 858 39

Tyrosine kinase growth factor receptors activate MAP kinase by a complex mechanism involving the SH2/3 protein Grb2, the exchange protein Sos, and Ras. The GTP-bound Ras protein binds to the Raf kinase and initiates a protein kinase cascade that leads to MAP kinase activation. Three MAP kinase kinase kinases have been described--c-Raf, c-Mos, and Mekk--that phosphorylate and activate Mek, the MAP kinase kinase. Activated Mek phosphorylates and activates MAP kinase. Subsequently, the activated MAP kinase translocates into the nucleus where many of the physiological targets of the MAP kinase signal transduction pathway are located. These substrates include transcription factors that are regulated by MAP kinase phosphorylation (e.g., Elk-1, c-Myc, c-Jun, c-Fos, and C/EBP beta). Thus the MAP kinase pathway represents a significant mechanism of signal transduction by growth factor receptors from the cell surface to the nucleus that results in the regulation of gene expression. Three MAP kinase homologs have been identified in the rat: Erk1, Erk2, and Erk3. Human MAP kinases that are similar to the rat Erk kinases have also been identified by molecular cloning. The human Erk1 protein kinase has been shown to be widely expressed as a 44-kDa protein in many tissues. The human Erk2 protein kinase is a 41-kDa protein that is expressed ubiquitously. In contrast, a human Erk3-related protein kinase has been found to be expressed at a high level only in heart muscle and brain. The loci of these MAP kinase genes are widely distributed within the human genome: erk2 at 22q11.2; erk1 at 16p11.2; and ek3-related at 18q12-21. In the yeast Saccharomyces cerevisiae, five MAP kinase gene homologs have been described: smkl, mpk1, hog1, fus3, and kss1. Together, these kinases are a more diverse group than the human erks that have been identified. Thus the erks are likely to represent only one subgroup of a larger human MAP kinase gene family. A candidate for this extended family of MAP kinases is the c-Jun NH2-terminal kinase (Jnk), which binds to and phosphorylates the transcription factor c-Jun at the activating sites Ser-63 and Ser-73. Evidence is presented here to demonstrate that Jnk is a distant relative of the MAP kinase group that is activated by dual phosphorylation at Tyr and Thr.
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PMID:Transcriptional regulation by MAP kinases. 860 77


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