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)

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.
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PMID:Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells. 239 35

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termed MMK2. The predicted amino acid sequence of MMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termed MMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness of MMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeast MPK1(SLT2) kinase by MMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.
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PMID:MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function. 747 71

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulates the activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase (MAPK), and S6 protein kinase (S6K) activities both in a time- and dose-dependent manner. A single peak of MAPKK activity, four peaks of activity against the S6 synthetic peptide, RRLSSLRA (S6 peptide), and three distinct peaks toward myelin basic protein (MBP) were observed after Mono-Q chromatography of LIF-stimulated cell extracts. Two of the MBP kinase activities correlated with the stimulation of extracellular signal-regulated kinases 1 and 2. Interestingly, down-regulation of protein kinase C (PKC) by chronic treatment of 3T3-L1 cells with phorbol ester was found to attenuate, but not block, the LIF-mediated stimulation of MAPKK, MAPK, and S6K activities in 3T3-L1 cells. Treatment of 3T3-L1 cells with epidermal growth factor increased MAPKK, MAPK, and S6K activities to a similar extent as LIF, but this activation was not attenuated by down-regulation of PKC. Our results suggest that the full activation of the MAPK cascade by LIF may require inputs from multiple signaling pathways, one of which is dependent upon the presence of functional PKC.
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PMID:Involvement of protein kinase C during activation of the mitogen-activated protein kinase cascade by leukemia inhibitory factor. Evidence for participation of multiple signaling pathways. 750 1

We have purified 42- and 44-kilodalton (kDa) isoforms of the mitogen-activated protein (MAP) kinase family from bovine brain. The kinases were assayed with myelin basic protein as the substrate and detected by anti-sea star p44mpk antibody. Purification was achieved using phenyl-Sepharose, polylysine-agarose, hydroxylapatite, and Mono-Q column chromatography. Both myelin basic protein and smooth muscle caldesmon, but not histone H1, served as good substrates. Based on chromatographic behaviors and specific activities toward myelin basic protein, it is likely that the 42-kDa brain isoform is similar to that of brain tau kinase. The 44-kDa enzyme, however, is a novel brain MAP kinase isoform not reported previously. Although it has been demonstrated that p44mpk can be activated in vitro through phosphorylation by the tyrosine kinase p56lck, neither of the brain kinases were significantly stimulated by the tyrosine kinases p56lck, p56lyn, or p59fyn. However, based on antibody cross-reactivity, a MAP kinase kinase is present in the crude brain extract. Both brain MAP kinases were capable of autophosphorylation which occurred, at least in part, on tyrosine residues. However, only the 44-kDa isoform showed a significant degree of coincident activation.
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PMID:MAP kinases from bovine brain: purification and characterization. 751 82

In this study, we examined the role of insulin, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) cascade in activation of protein phosphatase-1 (PP-1) by using three complementary approaches. First, differentiated L6 cells were acutely exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA, 400 nM) to activate PKC. In these cells, TPA caused 32% stimulation of PP-1 activity. The PP-1 stimulation by TPA was comparable to stimulation by insulin (t1/2 = 1 min and EC50 = 5 nM) with a maximum effect in 5 min. The effects of insulin and TPA were not additive. Insulin and TPA also stimulated MAPK (> 2-fold increase over basal, with myelin basic protein as a substrate). ML-9, a myosin light chain kinase inhibitor, blocked the effects of insulin and TPA on both MAPK and PP-1 activation. In the second approach, PKC was down-regulated by chronic treatment with TPA. In these cells subsequent effects of insulin on MAPK and PP-1 activation were blocked, without an effect on basal enzyme levels. In the third approach, two selective inhibitors of PKC, calphostin and chelerythrine chloride, were used to inhibit PKC. These inhibitors completely prevented insulin and TPA stimulation of MAPK and PP-1 and blocked insulin-induced translocation of PKC to the plasma membranes. We conclude that PKC plays an important role in insulin stimulation of PP-1 via the activation of MAPK cascade.
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PMID:Stimulation of protein phosphatase-1 activity by phorbol esters. Evaluation of the regulatory role of protein kinase C in insulin action. 751 82

Gamma interferon plays an important role in regulating the functional properties of mononuclear phagocytes. In the present study, the role of activated protein kinases in the mechanism of action of gamma interferon cell signaling in human peripheral blood monocytes was investigated. Analysis in vitro of 100,000 x g cytosolic fractions from untreated and interferon-treated cells showed that agonist treatment resulted in time- and concentration-dependent increases in phosphotransferase activity when myelin basic protein (MBP) was used as the substrate. Anion-exchange chromatography of high-speed supernatants prepared from detergent extracts of interferon-treated cells revealed two discrete peaks of MBP phosphotransferase activity. Immunoblotting of fractions from these peaks with antiphosphotyrosine antibodies and with antibodies that specifically recognize the family of mitogen-activated protein (MAP) kinases detected a MAP kinase with a subunit M(r) of 42,000 in the earliest-eluting peak (peak 1). Phosphorylation of the 42,000-M(r) protein on tyrosine was observed only after treatment of cells with interferon. The contribution of MAP kinase to the interferon-stimulated activity in peak 1 was confirmed by quantitative immunoprecipitation with anti-MAP kinase and antiphosphotyrosine antibodies. The conclusion that the interferon-activated MBP kinase in peak 1 could be accounted for by an activated MAP kinase was also supported by the finding that fractions from Mono Q peak 1 demonstrated activity towards a MAP kinase-specific substrate. The later-eluting peak of interferon-activated MBP phosphotransferase activity appeared to be accounted for by an activated protein kinase C (PKC). This conclusion is based upon analyses of immunoblotting and immunoprecipitation experiments with antibodies to PKC and was also supported by the observed inhibition of this kinase with a PKC pseudosubstrate peptide. The interferon-stimulated PKC present in Mono Q peak 2 was active in the absence of calcium ions, suggesting that it is a calcium-independent isoform of PKC.
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PMID:Gamma interferon induces rapid and coordinate activation of mitogen-activated protein kinase (extracellular signal-regulated kinase) and calcium-independent protein kinase C in human monocytes. 751 11

To investigate mechanisms of mononuclear phagocyte cell signaling, the effects of bacterial LPS on protein kinase activities in normal human peripheral blood monocytes were examined. Incubation of intact monocytes with LPS brought about time- and concentration-dependent increases in myelin basic protein (MBP) phosphotransferase activity in high speed supernatants of cell lysates. Anion-exchange chromatography on Mono Q demonstrated that LPS treatment resulted in two principal peaks of stimulated MBP kinase activity. Evidence was obtained to indicate that the first eluted peak of MBP kinase activity is accounted for by p42 and p44 mitogen-activated protein (MAP) kinases. Thus, 1) MBP kinase activity within peak 1 was quantitatively precipitated by anti-MAP kinase Abs, 2) the enzyme effectively phosphorylated a specific peptide substrate, 3) peak 1 contained proteins of subunit size M(r) 42,000 and M(r) 44,000 that reacted specifically with anti-MAP kinase Abs, and that 4) were recognized by anti-phosphotyrosine Abs only after stimulation of cells with LPS. Studies of the second peak of LPS-stimulated MBP kinase activity indicate that it is an isoform of protein kinase C (PKC) because: 1) enzyme activity was quantitatively immunoprecipitated by anti-PKC Abs, 2) the activity of the enzyme was potently and selectively inhibited by a specific peptide modeled on the autoinhibitory domain of PKC, and 3) the presence of a protein of subunit size M(r) 80,000 recognized by anti-PKC Abs. Because the second peak of MBP kinase activity (like the first) was active in the absence of added calcium and in the presence of 2 mM EGTA, it appears to be a type II, calcium-independent isoform of PKC. Abs to CD14 completely abrogated LPS-induced activation of both Mono Q peaks of MBP phosphotransferase activity. These results indicate that LPS coordinately activates both an apparently calcium-independent PKC and MAP kinase in mononuclear phagocytes and these responses appear to be initiated by signaling through the cell surface receptor, CD14.
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PMID:CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide. 752 66

Phosphorylation of the catalytic subunit of protein phosphatase 2A (PP2A) on threonines with a distinct autophosphorylation-activated protein kinase [Guo and Damuni (1993) Proc. Natl. Acad. Sci. USA 90, 2500-2504] inactivated the phosphatase with 32P-labelled myelin basic protein prepared by incubation with the kinase domain of the epidermal growth factor receptor, the src-family protein kinases p56lck and p60c-src, myelin basic protein kinase-1, or protamine kinase. Phosphoamino acid analysis demonstrated that the kinase domain of the epidermal growth factor receptor, p56lck and p60c-src phosphorylated myelin basic protein on tyrosines, that the protamine kinase phosphorylated myelin basic protein on serines, and that myelin basic protein kinase-1 phosphorylated myelin basic protein on threonines. The results demonstrate that the autophosphorylation-activated protein kinase not only inactivates the protein serine/threonine phosphatase, but also the protein tyrosine phosphatase activity of PP2A. This autophosphorylation-activated protein kinase-mediated inactivation of PP2A may, in response to extracellular stimuli, not only contribute to the enhanced phosphorylation of cellular proteins on serines and threonines but also on tyrosines.
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PMID:Autophosphorylation-activated protein kinase inactivates the protein tyrosine phosphatase activity of protein phosphatase 2A. 752 89

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 signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzymes.
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PMID:Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils. 752 47


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