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)

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.
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PMID:Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade. 786 10

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.
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PMID:Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS. 787 42

Previously pp60v-src, cyclin A, p39mos, and maturation-promoting factor (composed of Cdc2 and cyclin B) have been shown to activate mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) in cell-free extracts of Xenopus oocytes. The pp60v-src pathway is dependent on a functional Ras signal whereas the cyclin/maturation-promoting factor pathway is not. Here we show that protein kinase C (PKC) is also able to stimulate MAPK in a Ras-dependent manner, but PKC is not necessary for signaling by pp60v-src. In addition, preincubation of extracts with cAMP-dependent protein kinase (PKA) blocks stimulation of MAPK by cyclin, p21V12ras, PKC, or pp60v-src, by at least 50%, but stimulation by c-Mos is unaffected. Furthermore, inhibition of endogenous PKA by the heat-stable PKA inhibitor is sufficient to stimulate MAPK activity in these extracts in the absence of protein synthesis and without dependence on a functional Ras protein. These results suggest that independent pp60v-src and PKC pathways converge at Ras and that PKA acts to block MAPK activation by both Ras-dependent and -independent signals.
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PMID:Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system. 792 38

Recently, Sowadski and colleagues [Knighton, D.R., Zheng, J., Eyck, L.F.T., Ashford, V.A., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1991) Science 407, 407-420] reported the structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase (cyclic A kinase), MgATP and a 20-residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2-nm resolution and the orientation of the nucleotide and interactions of MgATP with numerous conserved residues at the active site defined [Zheng, J., Knighton, D.R., Eyck, L.F.T., Karlsson, R., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the alpha, beta and gamma phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [gamma-32P]adenosine-5'-(gamma-4-aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its gamma phosphate. In the presence of 20 microM ATP, adenosine-5'-(gamma-4-aminophenyl)triphosphate exhibited apparent Ki values of 103.6, 75.18, 176.28 and 120.00 microM against cyclic A kinase, mitogen-activated protein kinase (p42mapk), mitogen-activated protein kinase kinase and p60c-src, respectively. To illustrate the effectiveness of adenosine-5'-(gamma-4-aminophenyl)triphosphate-Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen-activated protein kinase kinase over 19000-fold to homogeneity.
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PMID:Gamma-phosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase. 851 96

The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family. Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock. It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants. We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation. Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants. In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase. We show here that wis1 function is required for mei2 induction following nitrogen starvation. Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function. Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function. These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets. The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1 delta genetic backgrounds.
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PMID:The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast. 883 15

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E2) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in 125I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 x g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E2 inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E2 abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E2 stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.
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PMID:Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cultured human luteinized granulosa cells. 894 64

In the fission yeast Schizosaccharomyces pombe, glucose represses onset of gluconate-H+ symport and inhibits transiently the activity of the symport protein. Wild-type cells harvested from high glucose medium take up gluconate very slowly and the rate of uptake is increased 150-fold in response to glucose starvation. Here it is shown that an intact cAMP cascade is necessary to prevent premature onset in the presence of high glucose concentrations. Cells which have lost either adenylate cyclase (Cyr1) or cAMP-dependent protein kinase (Pka1) transport gluconate up to 60-fold faster than wild-type cells when harvested from high glucose medium. Moreover, inactivation of the stress-sensing Wis1-Sty1 MAP kinase pathway, by loss of Wis1 MAP kinase kinase, diminishes 10-fold the onset of gluconate uptake in response to starvation. A mutant was identified showing a comparable phenotype. By complementation, the gti1+ (gluconate transport inducer 1) gene has been isolated. Disruption of gti1 reduces starvation-induced onset by a similar factor to that observed in wis1 delta cells. Cells over-expressing gti1+ induce gluconate uptake much faster resulting in a threefold higher uptake rate, although gti1+ does not code for the gluconate transport protein. In contrast to the repression of onset, transient downregulation of the gluconate symporter is independent of Pka1 activity and requires ongoing glucose influx. Addition of glucose to starved cyr1 delta cells reduces uptake 9-fold, whereas starved pka1 delta cells, which are able to synthesise cAMP, respond with a 60-fold decrease in transport.
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PMID:Onset of gluconate-H+ symport in Schizosaccharomyces pombe is regulated by the kinases Wis1 and Pka1, and requires the gti1+ gene product. 937 49

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.
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PMID:Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines. 938 11

To study the role of mitogen-activated protein kinase in the regulation of M2 receptors, we studied the effect of platelet-derived growth factor (PDGF) on M2 receptor gene expression. PDGF (4 ng/ml) caused a time-dependent decrease in M2 receptor number and in m2 receptor mRNA levels in HEL 299 cells. The PDGF-induced loss in m2 mRNA required de novo protein synthesis and occurred through a decrease in the rate of transcription of the m2 receptor gene. The down-regulation of M2 receptors was not accompanied by an uncoupling of the remaining receptors, indicating a large receptor reserve in these cells. Preincubations with the phosphatidylinositol 3-kinase inhibitor wortmannin, the protein kinase C inhibitor GF 109203X and the cAMP-dependent protein kinase inhibitor H-8 did not attenuate PDGF-induced down-regulation, indicating a lack of involvement of these enzymes in the down-regulation process. Activation of the extracellular signal-regulated protein kinase (ERK) 1 and 2 proteins was measured by an "in gel" phosphorylation assay. Carbachol did not activate ERK1 or 2, whereas PDGF and 4 beta-phorbol 13,14-dibutyrate resulted in a large increase in ERK1 and 2 activity along with a decrease in m2 mRNA. Preincubation with PD 098059, an inhibitor of mitogen-activated protein kinase kinase, inhibited PDGF- and 4 beta-phorbol 13,14-dibutyrate-mediated activation of ERK 1 and 2 in a concentration-dependent manner. The inhibitory action of PD 098059 was reflected at the mRNA level attenuating both PDGF- and 4 beta-phorbol 13,14-dibutyrate-mediated decreases in m2 mRNA. These results suggest a role of ERK1 and 2 in the regulation of muscarinic m2 receptor gene expression.
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PMID:Regulation of m2 muscarinic receptor gene expression by platelet-derived growth factor: involvement of extracellular signal-regulated protein kinases in the down-regulation process. 941 6

Glutamate and dopamine are important neurotransmitters in the basal ganglia. Dopamine can act via D1 receptors to activate adenylyl cyclase in striatal neurons, while glutamate stimulation of NMDA receptors leads to an increase in intracellular calcium. Increases in intracellular calcium or cAMP can induce immediate early gene expression in striatal neurons. In the present study, NMDA receptor stimulation or adenylyl cyclase activation resulted in the activation of MAP kinase in striatal neurons in primary culture. The effect of cAMP appeared to involve cAMP-dependent protein kinase, in addition to a tyrosine kinase and MEK. NMDA-induced MAP kinase activation was also dependent on a tyrosine kinase and MEK. The EGF receptor, which has been implicated in calcium- and G protein-induced MAP kinase activation, did not mediate the effects of NMDA or forskolin on MAP kinase. Furthermore, the src kinase inhibitor, herbimycin A, and the phosphoinositol-3-kinase inhibitor, wortmannin, did not prevent MAP kinase activation by these stimuli. However, the ability of both NMDA and forskolin to activate MAP kinase in striatal neurons was blocked by SB 203580, an inhibitor of p38 reactivating kinase. These results indicate that both NMDA receptor activation and elevations in cAMP can result in MEK-induced MAP kinase activation in striatal neurons. However, the signal transduction pathways mediating these responses appear to be distinct from those known to mediate MAP kinase activation by other stimuli.
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PMID:Neurotransmitter regulation of MAP kinase signaling in striatal neurons in primary culture. 955 73


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