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:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of insulin to its receptor initiates multiple signal transduction pathways regulating such diverse processes as proliferation, differentiation, glucose transport, and glycogen metabolism. The STAT-family of transcription factors has been demonstrated to play a critical role in gene induction by a variety of hemopoietic cytokines and hormones. Furthermore, constitutive activation of STATs is observed in transformed cells. Here we describe activation of a transcriptional complex binding to a consensus STAT-transcriptional element in response to insulin challenge. This complex is induced rapidly after tyrosine autophosphorylation of the insulin receptor, and is sustained for several hours. Supershift analysis of the insulin-induced complex reveals that it specifically contains the transcription factor Stat3. DAN binding of this complex is inhibited by pre-incubation with tyrosine, but not serine/threonine protein kinase inhibitors, whereas transcriptional activation is inhibited by both. Utilising a dominant negative mutant of p21ras we demonstrate that both insulin-induced Stat3 DNA-binding and also transactivation do not require p21ras. Furthermore, although previous studies have suggested a role for MAP kinases (ERKs) and PI-3K in STAT activation, utilising the specific MEK inhibitor PD098059 and the PI-3K inhibitor wortmannin, we demonstrate that activation of ERKs or PI-3K are not required for insulin induced Stat3 phosphorylation or transactivation.
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PMID:Insulin activates Stat3 independently of p21ras-ERK and PI-3K signal transduction. 939 41

We investigated subcellular distribution of ERK2 in osteoblast-like UMR-106 cell and explored to determine if its activities are regulated by insulin. 23%, 34% and 43% of total ERK2 were distributed in membrane, cytosol and nucleus, respectively. Insulin caused 40% increase of ERK2 content in membrane in 10 min whereas it induced approximately 50% decrease of ERK2 in cytosol in 10 min. In terms of kinase activity, insulin stimulated phosphorylation of the membrane-associated ERK2 by 2-fold and 1.8-fold in 1 min and 10 min and cytosolic ERK2 by 2.7-fold and 2.3-fold in 1 min and 10 min, respectively. In contrast, the phosphorylation of nuclear ERK2 was stimulated by insulin in time-dependent manner with maximal (3-fold) activity observed at 30 min. Insulin also increased the content of MEK2 in membrane by 2.2- to 2.6-fold in 10 min. MEK2 translocated into membrane in response to insulin may play a role in the activation of the membrane-associated ERK2 via phosphorylation.
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PMID:Insulin rapidly stimulates ERK2 in the membrane of osteoblast-like UMR-106 cell. 941 11

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.
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PMID:Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase. 942 18

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect. Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB. In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf --> MEK pathway and has an impact on physiologically relevant genes in these cells.
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PMID:Insulin stimulates cAMP-response element binding protein activity in HepG2 and 3T3-L1 cell lines. 942 50

The insulin-stimulated uptake of 2-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable substrate for system A, in 3T3-L1 adipocytes was investigated. As cells took on a more adipogenic phenotype, the insulin-stimulated versus the saturable basal MeAIB uptake increased by 5-fold. The induced transport activity showed properties characteristic of system A, with a Km value of 190 microM. The half-life of the induced system A activity was independent of de novo mRNA and protein synthesis and was not accelerated by ambient amino acids, therefore, it was mechanistically distinct from the previously described adaptive and hormonal regulation of system A. Inhibition of mitogen-activated protein kinase kinase by PD98059, Ras farnesylation by PD152440 and B581, p70(S6K) by rapamycin, and phosphatidylinositol 3-kinase (PI 3'-K) by wortmannin and LY294002 revealed that only wortmannin and LY294002 inhibited the insulin-induced MeAIB uptake with IC50 values close to that previously reported for inhibition of PI 3'-K. These results suggest that the Ras/mitogen-activated protein kinase and pp70(S6K) insulin signaling pathways are neither required nor sufficient for insulin stimulation of MeAIB uptake, and activation of PI 3'-K or a wortmannin/LY294002-sensitive pathway may play an important role in regulation of system A transport by insulin in 3T3-L1 cells.
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PMID:Regulation of system A amino acid transport in 3T3-L1 adipocytes by insulin. 945 28

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the first committed step in hepatic gluconeogenesis. Glucagon and glucocorticoids stimulate PEPCK gene transcription, whereas insulin has a dominant inhibitory effect. We have shown that inhibitors of 1-phosphatidylinositol 3-kinase (PI 3-kinase) block this action of insulin. In contrast, three distinct agents, all of which prevent activation of p42/p44 mitogen-activated protein (MAP) kinase, have no effect on the regulation of PEPCK transcription by insulin. However, a subsequent report has suggested that this pathway is involved in the inhibition of cAMP-induced PEPCK gene transcription by insulin. To address these conflicting data, we re-examined the Ras MAP kinase pathway, not only with respect to regulation of PEPCK gene transcription, but also for regulation of PI 3-kinase and p42/p44 MAP kinase. Overexpression of constitutively active Ras (V61) (or Raf-1 (RafCAAX)) partially represses PEPCK transcription in hepatoma cells. However, an inhibitor of MAP kinase kinase blocks this action of RafCAAX but has no effect on regulation of PEPCK gene transcription by insulin. Second, the action of a dominant negative Ras (N17Ras) on PEPCK gene transcription correlates more closely with the inhibition of PI 3-kinase than with the inhibition of p42/p44 MAP kinase. Third, insulin cannot activate p42/p44 MAP kinase in the presence of cAMP even though cAMP-induced PEPCK gene transcription is inhibited by insulin. This data confirms that the Ras MAP kinase pathway is not required for the regulation of PEPCK gene transcription by insulin and demonstrates the importance of employing multiple techniques when investigating the function of signaling pathways.
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PMID:Activation of the ras mitogen-activated protein kinase-ribosomal protein kinase pathway is not required for the repression of phosphoenolpyruvate carboxykinase gene transcription by insulin. 945 31

In MCF7 breast cancer cells, mitogen-activated protein (MAP) kinase (i.e. Erk-1/2) is activated by the mitogen insulin, but also by the growth inhibiting agent TPA, though with very different kinetics. Insulin induces a relatively transient activation of Erk2 (<15 min), whereas TPA is able to induce a prolonged activation of Erk2 (>6 h). Expression of immediate-early genes of the c-fos and c-jun families, whose transcription and activation are regulated by MAP kinases, is differentially induced by insulin and TPA. Whereas insulin stimulates prolonged induction of c-jun, but not of junB mRNA, resulting in c-jun expression during the entire G1 period, the growth inhibitor TPA induces junB much longer than c-jun. Inhibition of the Erk2 pathway by PD98059, specific for the upstream MAP kinase kinase (MEK1), abolishes TPA-stimulated junB but not insulin-induced c-jun. In agreement with this, insulin readily stimulates Jun kinase (JNK), whereas TPA does not. Furthermore, insulin-induced pRB hyperphosphorylation at the G1-S transition and S-phase entry is insensitive to MAP kinase inhibition by PD98059. On the other hand, PD98059 reverts the inhibitory effect of TPA on cell cycle entry as well as on pRB hyperphosphorylation, indicating that Erk effectors function as inhibitors of proliferation in MCF7 cells.
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PMID:The role of MAP kinase in TPA-mediated cell cycle arrest of human breast cancer cells. 946 52

In hepatocytes glycogen storage is stimulated by insulin and this effect of insulin is counteracted by epidermal growth factor (EGF). The mechanism by which insulin stimulates glycogen synthesis in liver is unknown. We investigated the involvement of candidate protein kinases in insulin signalling in hepatocytes. Both insulin and EGF activated extracellular regulated kinase 2 (ERK-2), p70rsk and protein kinase B (PKB) and inactivated glycogen synthase kinase-3 (GSK-3). Whereas EGF caused a greater activation of ERK-2 than insulin, the converse was true for PKB. The stimulation by insulin of ERK-2 was blocked by a mitogen-activated protein (MEK) inhibitor (PD 98059) and of p70rsk by rapamycin. However, these inhibitors, separately or in combination, did not block the stimulation of glycogen synthesis by insulin, indicating that activation of these kinases is not essential for the stimulation of glycogen synthesis by insulin. Mono Q fractionation of hepatocyte extracts resolved a single myelin basic protein (MBP) kinase peak from extracts of EGF-treated cells (peak 1, eluting at 200 mmol/l NaCl) and two peaks from insulin-treated cells (peak 1 eluting at 200 mmol/l NaCl and peak 2 eluting at 400 mmol/l NaCl). In the combined presence of insulin and EGF, activation of peak 2 was abolished. In situ MBP kinase assays and immunoblotting established that peak 1 coincides with ERK-2 and peak 2 is not an activated form of ERK-1 or ERK-2. It is concluded that PKB, which is activated to a greater extent by insulin than EGF, and peak 2, which is activated by insulin and counteracted by EGF, are possible candidates in mediating the stimulation of glycogen synthesis by insulin.
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PMID:Signalling pathways involved in the stimulation of glycogen synthesis by insulin in rat hepatocytes. 949 25

Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-betaB5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-betaB7 affects binding only to the receptors, while a mutation in Thr-betaC5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.
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PMID:Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases. 955 7

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


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