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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)

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.
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PMID:Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. 749 78

Site directed mutagenesis/charged-to-alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione-S-transferase fusion protein (GST-ERK2) from E. coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (-9aa[40-48]) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140-240% but with kinase activity toward MBP ranging from 50-100% of the wild type.
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PMID:Isolation and characterization of mutants of human mitogen-activated protein kinase (ERK2). 752 93

The 21 kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. Less well understood are the downstream signal transduction cascades initiated by Ras in response to external stimuli. Only recently have many diverse studies in lower eukaryotes and vertebrates converged to demonstrate that Ras directly regulates multiple signaling pathways. In most eukaryotes, Ras functions as a positive regulator of an ERK/MAPK signal transduction cascade through the activation of a MEKK. In mammalian cells the primary Ras-responsive MEKK is the protein kinase Raf. Although Raf remains the most significant mediator of Ras signaling in most model systems, it does not explain all the biochemical responses observed in cells with activated Ras proteins. Yeast two hybrid and GST-fusion protein binding studies have identified new proteins distinct from Raf that could interact with Ras in other signaling pathways. In addition to Raf, other potential Ras target proteins include MEKK1, PI(3)K, p120GAP, ralGDS, and PKC zeta. This review will attempt to summarize the current literature of accepted and potential Ras-dependent signaling proteins in both lower eukaryotes and vertebrates.
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PMID:Ras target proteins in eukaryotic cells. 755 21

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.
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PMID:A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase. 778 77

MAPK-activated protein kinase-2 (MAPKAP kinase-2) is activated in vitro by the p42 and p44 isoforms of MAPK (p42/p44MAPK). In several cell lines, however, MAPKAP kinase-2 is activated by sodium arsenite, heat shock, or osmotic stress and not by agonists that activate p42/p44MAPK. We have identified a MAPK-like enzyme that acts as a MAPKAP kinase-2 reactivating kinase (RK). RK is recognized by an antiserum raised against a Xenopus MAPK (Mpk2), which is most similar to HOG1 from S. cerevisiae. We also identified a RK kinase (RKK) on the basis of its ability to activate either RK or a GST-Mpk2 fusion protein. The RKK, RK, and MAPKAP kinase-2 constitute a new stress-activated signal transduction pathway in vertebrates that is distinct from the classical MAPK cascade.
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PMID:A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. 792 53

Mitogen activated protein kinases (MAP) or extracellular signal regulated protein kinases (ERK) are a family of protein serine/threonine kinases that are activated very rapidly in response to many extracellular stimuli. elk-1, an ets related gene codes for two transcriptional factors elk-1, which regulates c-fos transcription and delta elk-1, both of which are substrates for MAP kinases. A part of the C-terminal transcriptional activation domain (ETA-2) which is common to both the proteins was previously shown to function as an activator of MAP kinases. In this report, in an attempt to investigate the mechanism of activation of MAP kinases, purified preparations of recombinant elk-1 and P44mpk/ERK-1/ERK-2 proteins were used to show the association of elk-1 proteins with MAP kinases. The specific interactions of elk-1 proteins with MAP kinases were confirmed by co-immunoprecipitation studies. Thus elk-1 proteins appear to regulate the activity of MAP kinases by interacting with them ensuring a conformational change and stimulating their autophosphorylation and activation property. The activation was dependent on the presence of ATP and Mg2+. In vitro phosphorylation of elk-1 protein was not regulatory for autonomous DNA binding activity of elk-1 protein. Cells which were exposed to EGF showed a rapid stimulation of an elk-1 specific kinase activity, probably MAP kinase which phosphorylated MBP and was found to be associated with immobilized GST-elk-1. Furthermore, dephosphorylation studies indicate that elk-1 proteins can activate only tyrosine phosphorylated MAP kinase. These results demonstrate the presence of an alternative pathway/mechanism (other than MAP kinase kinase, MAPKK/Mek) for the activation of MAP kinases with tyrosine phosphorylation occurring before serine/threonine autophosphorylation and activation by elk-1 proteins.
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PMID:elk-1 proteins interact with MAP kinases. 820 31

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.
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PMID:Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes. 852 17

The effects of EGF, TPA, UV radiation, okadaic acid and anisomycin on ERK and JNK/SAPK MAP kinase cascades have been compared with their ability to elicit histone H3/HMG-14 phosphorylation and induce c-fos and c-jun in C3H 10T1/2 cells. EGF and UV radiation activate both ERKs and JNK/SAPKs but to markedly different extents; EGF activates ERKs more strongly than JNK/SAPKs, whereas UV radiation activates JNK/SAPKs much more strongly than ERKs. Anisomycin and okadaic acid activate JNK/SAPKs but not ERKs, and conversely, TPA activates ERKs but not JNK/SAPKs. Nevertheless, all these agents elicit phosphorylation of ribosomal and pre-ribosomal S6, histone H3 and HMG-14, and the induction of c-fos and c-jun, showing that neither cascade is absolutely essential for these responses. We then analysed the relationship between ERKs, JNK/SAPKs and the transcription factors Elk-1 and c-Jun, implicated in controlling c-fos and c-jun, respectively. JNK/SAPKs bind to GST-cJun1-79, and ERKs, particularly ERK-2, to GST-Elk1(307-428); there is no cross-specificity of binding. Further, GST-Elk1(307-428) binds preferentially to active rather than inactive ERK-2. In vitro, JNK/SAPKs phosphorylate both GST-cJun1-79 and GST-Elk1(307-428), whereas ERKs phosphorylate GST-Elk1(307-428) but not GST-cJun1-79. Thus, neither ERKs nor JNK/SAPKs are absolutely essential for nuclear signalling and c-fos and c-jun induction. The data suggest either that activation of a single MAP kinase subtype is sufficient to elicit a complete nuclear response, or that other uncharacterised routes exist.
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PMID:Neither ERK nor JNK/SAPK MAP kinase subtypes are essential for histone H3/HMG-14 phosphorylation or c-fos and c-jun induction. 858 71

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.
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PMID:Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. 862 50

Bacterial LPS stimulation of murine macrophages leads to increased tyrosine phosphorylation and activation of the 42- and 44-kDa mitogen-activated protein kinases (MAPK) and the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) and p38, related to the high osmolarity glycerol protein kinase in Saccharomyces cerevisiae (HOG1). LPS caused a rapid increase (10 min) in phosphotransferase activity toward myelin basic protein (MBP), a polypeptide that encompassed the first 169 residues of c-Jun fused to gluthathione S-transferase (GST-c-Jun (1-169)) and 27-kDa heat shock protein (hsp27). MonoQ fractionation of cell extracts resolved phosphotransferase activity peaks toward MBP, GST-c-Jun (1-169), and hsp27, which contained MAPK, SAPK/JNK, and MAPKAPK2, respectively, as indicated by immunoblotting data. In RAW 264.7 macrophages, LPS stimulation of MAPKAPK2, a substrate of p38 HOG1 and MAPK, appeared to occur predominantly via p38 HOG1 and not the MAPK. PMA, which activated the MAPK as potently as LPS, did not strongly activate MAPKAPK2, as assessed by hsp27 phosphorylation. Consistent with p38 HOG1-mediating LPS activation of MAPKAPK2, treatment with LPS, but not PMA, increased the tyrosine phosphorylation of p38 HOG1, a modification known to elevate the enzymatic capacity of this kinase. In LPS-treated cells, the activity of SAPK/JNK was increased 5- to 10-fold, as measured by precipitating SAPK/JNK with Abs or immobilized GST-c-Jun and performing an in vitro kinase assay. In addition, the kinases thought to be upstream of SAPK/JNK, SAPK/ERK kinase 1 (SEK1), and MAPK/ERK kinase kinase 1 (MEKK1), were activated following LPS, but not PMA, exposure (5-fold and 2.5-fold, respectively.
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PMID:Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages. 866 21


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