Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.25 (MEKK1)
 1,856 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 microM) inhibited the activation of p42MAPK and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented. These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway.
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PMID:PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. 749 6

Numerous potential activators of MEK have been identified, including c-Raf-1, B-Raf, c-Mos, and a family of MEK kinases. However, little information gives insight into the activators actually utilized in vivo. To address this, we have used column chromatography and a coupled MEK activation assay to identify in NIH3T3 cells, two major MEK activators, and a third insulin-specific activator. The first MEK activator has an apparent M(r) of 40,000-50,000, was immunologically distinct from A-Raf, B-Raf, c-Raf-1, c-MEKK, c-Mos, MEK1, and MEK2, and was rapidly activated by serum, platelet-derived growth factor (PDGF), insulin, thrombin, and phorbol ester. The second MEK activator was identified as B-Raf. Activation of 93-95 kDa B-Raf was observed in column fractions and B-Raf immunoprecipitates from cytosolic and particulate fractions after stimulation with serum or PDGF, but not insulin. c-Raf-1 from cytosol did not exhibit MEK activator activity; however, c-Raf-1 immunoprecipitates from the particulate fraction revealed MEK activator activity that was enhanced after stimulation with PDGF or phorbol ester, but not serum or insulin. Both c-Mos and c-MEKK were present in NIH3T3 fibroblasts but did not show MEK activator activity. These data provide direct evidence that 93-95-kDa B-Raf isozymes and an unidentified 40-50-kDa MEK activator are major agonist-specific MEK activators in NIH3T3 fibroblasts.
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PMID:Biochemical analysis of MEK activation in NIH3T3 fibroblasts. Identification of B-Raf and other activators. 770 12

Treatment of adipocytes with insulin or phorbol 12-myristate 13-acetate (PMA) results in transient activation of mitogen-activated protein kinase kinase (MEK) (Tmax = 90 s) and mitogen-activated protein kinase (MAPK) (Tmax = 300 s). We have identified a novel insulin-stimulated MEK kinase (I-MEKK) in the 100,000 x g infranatant that shows rapid phasic kinetics that temporally precede that of MEK. Maximal activation of I-MEKK occurs within 20 +/- 5 s (S.D., n = 3) followed by complete inactivation by 30 +/- 10 s (S.D., n = 3). I-MEKK was characterized by anion-exchange and gel filtration chromatography and separated into two distinct activities of approximately 56 kDa that phosphorylated and activated MEK. I-MEKKs did not co-elute on anion exchange with c-Raf or 73-kDa MEK kinase (MEKK), suggesting they are distinct enzymes. Protein phosphatase 2A inactivated both I-MEKKs in vitro and in the intact cell okadaic acid blocked inactivation in the presence of insulin. These results suggest activation of I-MEKK involves phosphorylation on serine or threonine residues. I-MEKK was not activated by PMA, suggesting that in adipocytes the enzyme represents a divergence point between signal transduction pathways mediated by insulin and those activating protein kinase C.
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PMID:Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf. 817 93

Recently, it has been reported that Raf-1 kinase (Raf-1) has mitogen-activated protein kinase kinase kinase (MAPKKK) activity in various cells, although Raf-1 and MAP kinase kinase (MAPKK) can be phosphorylated by MAP kinase (MAPK) in vitro. Here we show that the maximal hyperphosphorylation of Raf-1 and MAPKK (10 min) was substantially achieved after the maximal activation of MAPKKK of Raf-1, MAPKK (2-5 min), and MAPK in Chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) treated with insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA). Moreover, we show that overexpression of MAPK in CHO-HIR cells resulted in enhanced hyperphosphorylation of Raf-1, MAPKK, and mammalian homolog of son of sevenless (mSos) after insulin or TPA stimulation as compared with parental cells. Furthermore, the maximal hyperphosphorylation of Raf-1 appears to be accompanied by a significant decrease in MAPKKK activity. These results suggest that 1) signals initiated by insulin and TPA converge on Raf-1 and activate its MAPKKK activity and 2) Raf-1, MAPKK, and mSos not only lie upstream of MAPK but also are phosphorylated by MAPK, directly or indirectly, and at least Raf-1 kinase activity might be down-regulated by this feedback mechanism.
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PMID:Feedback regulation of mitogen-activated protein kinase kinase kinase activity of c-Raf-1 by insulin and phorbol ester stimulation. 819 29

cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.
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PMID:A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity. 966 34

The aim of this study was to define the role of sterol regulatory element binding protein (SREBP)-1c, the human homologue to ADD1 (adipocyte determination- and differentiation-dependent factor 1), in insulin-induced gene expression. Transfection studies using SREBP-1-deficient cells and a LDL receptor promoter fragment containing the ADD1/SREBP-1c binding side showed that the effects of insulin and PDGF were abolished compared to control cells and completely reconstituted by overexpressing ADD1/SREBP-1c. Overexpression of upstream activators of MAP kinases, like MEKK1 or MEK1, demonstrated that ADD1/SREBP-1c-mediated effects of insulin and PDGF might be linked to the MAP kinase cascade. The recombinant N-terminal domain of ADD1/SREBP-1c was phosphorylated predominantly on serine and slightly on threonine residues by MAP kinases ERK1 and ERK2 in vitro. This was reversible by alkaline phosphatase. We conclude that ADD1/SREBP-1c mediates gene regulatory effects of insulin as well as PDGF and that this signalling is linked to the MAP kinase cascade.
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PMID:ADD1/SREBP-1c mediates insulin-induced gene expression linked to the MAP kinase pathway. 971 4

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.
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PMID:Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis. 1037 46

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.
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PMID:Increased tyrosine kinase activity but not calcium mobilization is required for ceramide-induced apoptosis. 1041 87

The classic sterol regulatory cis element (sre-1) in the LDL receptor promoter mediates sterol regulatory element binding protein (SREBP)-binding and the effects of insulin and platelet derived growth factor (PDGF). To elucidate whether SREBP-1a and SREBP-2 play a direct role in insulin and PDGF action, stable cell lines of HepG2 deficient in either SREBP-1 or SREBP-2 were used. Transfection of these cells with the wild-type promoter fragment of the low density lipoprotein (LDL) receptor gene showed that the effects of insulin and PDGF were significantly reduced in both, SREBP-1- as well as SREBP-2-deficient cells. Insulin and PDGF action could be reconstituted again in these deficient cell lines by reintroducing SREBP-1a or SREBP-2. Preincubation of cells with either the phosphatidylinositol (PI)-3 kinase inhibitor wortmannin or the mitogen-activated protein (MAP) kinase cascade inhibitor PD 98059 showed that the latter abolished the stimulatory effects of insulin and PDGF on LDL receptor promoter activity completely, whereas wortmannin had no effect. Overexpression of upstream activators of the MAP kinases, like MEKK1 or MEK1, stimulated LDL receptor promoter activity several fold in an sre-1 related manner. These effects could be enhanced by coexpression of the transcriptional active N-terminal domains of SREBP-1a and SREBP-2. Using the heterologous Gal-4 system, we could show that intracellular activation of the MAP kinase cascade by ectopic expression of MEKK1 or MEK1 has a direct stimulatory effect on the transcriptional activity of SREBP-1a and SREBP-2. Experimental evidence for a direct link between MAP kinases and SREBPs was obtained due to the MAP kinases ERK1 and ERK2 phosphorylating recombinant GST-fusion proteins of SREBP-1a and SREBP-2, in vitro. We conclude that SREBP-1a and SREBP-2 mediate different regulatory effects converging at sre-1 and that they appear to be linked to the MAP kinase cascade, possibly being direct substrates of ERK1 and ERK2.
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PMID:Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP-kinase cascade. 1062 7

The protein serine-threonine kinase Akt mediates cell survival signaling initiated by various growth-promoting factors such as insulin. Here we report that SEK1 is a target of Akt in intact cells. Insulin inhibited the anisomycin-induced stimulation of both endogenous SEK1 and its substrate c-Jun N-terminal kinase (JNK), but not that of the upstream kinase MEKK1, in 293T cells. The inhibitory action of insulin on SEK1 or JNK1 activation was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002. Expression of a constitutively active form of Akt also inhibited both SEK1 and JNK1 activation, but not that of MEKK1, in transfected 293T cells. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacted with endogenous SEK1 in cells and that this interaction was promoted by insulin. In vitro and in vivo (32)P labeling indicated that Akt phosphorylated SEK1 on serine 78. The SEK1 mutant SEK1(S78A) was resistant to Akt-induced inhibition. Finally, activated Akt inhibited SEK1-mediated apoptosis, and this effect of Akt was prevented by overexpression of SEK(S78A). Taken together, these results suggest that Akt suppresses stress-activated signaling by targeting SEK1.
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PMID:Akt (protein kinase B) negatively regulates SEK1 by means of protein phosphorylation. 1170 64


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