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:2.7.11.1 (protein kinase)
81,284 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

Chymotryptic digestion was used to localize the sites in microtubule-associated protein 2 which are preferentially phosphorylated in vitro by MAP kinase, an insulin-stimulated serine/threonine kinase which efficiently utilizes high molecular weight MAPs as substrates. MAP kinase phosphorylates sites in the projection domain almost exclusively; less than 6% of the phosphate incorporated by MAP kinase was found in the tubulin binding domain. This site specificity is in marked contrast to that of the catalytic subunit of cAMP dependent protein kinase, and most other protein kinases phosphorylating MAP-2, which extensively phosphorylate the tubulin binding domain.
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PMID:Phosphorylation of microtubule-associated protein 2 by MAP kinase primarily involves the projection domain. 254 2

pp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from G0 into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase II. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase". We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one- and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.
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PMID:Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. 255 Sep 26

A protein kinase, termed microtubule-associated protein (MAP) kinase, which phosphorylates microtubule-associated protein 2 (MAP-2) in vitro and is stimulated 1.5-3-fold in extracts from insulin-treated 3T3-L1 cells has been identified (Ray, L.B., and Sturgill, T.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1502-1506). Here, we describe chromatographic properties of MAP kinase and provide biochemical characterization of the partially purified enzyme. Isolation of the enzyme is facilitated by its unusually high affinity for hydrophobic interaction chromatography matrices. The molecular weight of the partially purified enzyme was determined to be 35,000 by gel filtration chromatography and 37,000 by glycerol gradient centrifugation. MAP kinase activity of chromatographic fractions correlated precisely with the presence of a 40-kDa phosphoprotein detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAP kinase has a Km of 7 microM for ATP and does not utilize GTP. Acetyl-CoA carboxylase, ATP citrate-lyase, casein, histones, phosvitin, protamine, and ribosomal protein S6 were all poor substrates relative to MAP-2. The enzyme is inhibited by fluoride and beta-glycerol phosphate but not by heparin. These properties of MAP kinase distinguish it from protein kinases previously described in the literature.
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PMID:Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells. 284 41

Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with 32Pi. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The 32P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extent phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. We conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.
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PMID:Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. 328 75

We investigated the effects of epidermal growth factor (EGF) and arginine vasopressin (AVP) on Raf-1-MAP kinase cascade, including Raf-1-kinase (Raf-1-K), MAP kinase kinase (MAPKK), MAP kinase (MAPK) and S6 kinase (S6K) in Madin-Darby canine kidney (MDCK) cells. In a dose-dependent manner (10(-10) M to 10(-6) M), EGF increased autophosphorylation of Raf-1-K and activated MAPKK, MAPK and S6K. Sequential activation of these kinases was indicated by their peak times of activation (Raf-1-K 5 min; MAPKK 10 min; MAPK 15 min; and S6K 30 min). AVP (10(-9) M to 10(-6) M) inhibited EGF-stimulated MAP kinase cascade. 8-Bromo-cyclic AMP (cAMP) could mimic the inhibitory effect of AVP on EGF-stimulated MAP kinase cascade. These results were confirmed using H-89, an inhibitor of protein kinase A (PKA) that blocked the effect of AVP on EGF-stimulated MAPK activity. We conclude that AVP inhibits EGF-stimulated Raf-1-K, MAPKK, MAPK, and S6K activity via cAMP in MDCK cells. Our results indicate that MAP kinase cascade may play an important role in integrating the effects of AVP and EGF on distal tubule function.
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PMID:AVP inhibits EGF-stimulated MAP kinase cascade in Madin-Darby canine kidney cells. 747 60

The MAP kinase pathway impinging on ERK2 has been shown to be integrally associated with mitogenic signalling in many cell types. Previously, we and others have demonstrated that oncogenic forms of Raf-1 kinase, when expressed in fibroblasts, lead to the constitutive activation of ERK2, the de-regulation of c-fos expression and increased cell proliferation. Here we describe an exception to this scenario. In Rat6 cells, although both ERK1 and ERK2 are activated in response to mitogens that induce c-fos expression, such as Epidermal Growth Factor (EGF), lysophosphatidic acid (LPA) or serum, expression of v-Raf fails to induce c-fos expression and increase proliferation. However, ERK2 is activated by v-Raf expression. The co-transfection of an interfering mutant of ERK2 has no effect on the level of c-fos reporter expression in Rat6 cells whereas the analogous ERK1 mutant reduces its expression. Furthermore, the spontaneous focus formation observed in Rat6 cells is susceptible to the interfering mutant of ERK1 but resistant to that of ERK2. Thus, not only do mitogenic signals appear to by-pass both Raf-1 kinase and ERK2, the Raf-1-ERK2 pathway seems to be functionally compromised in Rat6 cells as its activation leads neither to c-fos expression nor to increased proliferation.
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PMID:Raf-1 kinase and ERK2 uncoupled from mitogenic signals in rat fibroblasts. 747 30

Activation of the mitogen-activated protein (MAP) kinase pathway is believed to play a critical role in normal and pathophysiological proliferation of mesangial cells. Recent studies have shown that MAP kinase activation by growth factors in other cell types involves activation of the low-molecular-weight G protein Ras and the protooncogene serine kinase c-Raf-1. In this study, the role of this pathway in rat renal mesangial cells was assessed. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), as well as phorbol esters (PMA) rapidly activated MAP kinase three- to fourfold in these cells. PDGF and EGF, but not PMA, were able to activate c-Raf-1 and Ras activity. Stimulation of mesangial cells with the inflammatory mediator prostaglandin E2 (PGE2) or elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by treatment with forskolin markedly blunted activation of MAP kinase induced by PDGF and EGF, but not by PMA. Consistent with this observation, PGE2 abolished growth factor-induced activation of c-Raf-1. However, Ras activation induced by growth factors was not affected by PGE2 and forskolin. These results suggest that MAP kinase activation can occur by at least two separate pathways in mesangial cells. Tyrosine kinase receptors activate MAP kinase through activation of Ras and Raf. This pathway can be blocked by PGE2 and elevation of cAMP, presumably by interfering with the ability of Ras to activate Raf. In addition, activation of protein kinase C by phorbol esters can activate MAP kinase in a Ras/Raf-independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of MAP kinase by prostaglandin E2 and forskolin in rat renal mesangial cells. 748 69

Figure 2 summarizes our current interpretation of data concerning signals from the activated PDGF receptor involved in directed migration and proliferation of human arterial SMC. Binding of PDGF (PDGF-BB or PDGF-AA) causes PDGF-receptor dimerization, tyrosine autophosphorylation, and subsequent binding of several molecules containing SH2 domains to the activated receptor. Binding and activation of PLC gamma by the PDGF receptor leads to PIP2 hydrolysis, resulting in generation of diacylglycerol (DAG) and IP3. Subsequently, intracellular levels of calcium are elevated as a result of IP3-mediated calcium release from intracellular compartments. The decreased levels of PIP2 and increased levels of calcium both favor actin-filament disassembly by inducing capping of actin-filament barbed ends and actin-monomer sequestration. A localized, and transient, actin-filament disassembly enables the cell to extend filopodia towards PDGF, thereby enabling chemotaxis to take place. At a later time and/or in a different compartment, actin-filament assembly is promoted by PDGF by a mechanism that is not completely understood, but that may involve small GTP-binding proteins, such as Rho, and formation of DAG. Migration on collagen requires functional alpha 2 beta 1 integrins, which may either constitute a permissive state required for a cell to migrate, or which may be actively involved in intracellular signals leading to migration. PDGF-induced DNA synthesis and proliferation involves activation of Ras, MAP kinase kinase, and MAP kinase. Cross-talk between PKA signaling and tyrosine-kinase receptor signaling results in PKA inhibition of the MAP kinase cascade, probably at the level of Raf. Activation of PI 3-kinase, or a PI 3-kinase-like enzyme, is also likely to contribute to the mitogenic effects of PDGF in these cells (Bornfeldt, unpublished observation). What determines if a SMC will migrate and/or proliferate in response to PDGF? Results are starting to emerge that show regulation of expression of molecules involved in intracellular signaling with different phenotypic states of SMC. For example, expression of PLC gamma is very low in intact vascular wall (where SMC show a "contractile phenotype"), and induced when SMC are converted to a "synthetic phenotype" in culture. Proliferation and expression of MAP kinase, but not calcium signaling, appear to be regulated by the extracellular matrix, and the profile of integrin expression is different in SMC in culture compared to SMC in the vascular wall. Thus, the relation between expression of signaling molecules involved in migration and signaling molecules involved in proliferation, as well as cross-talk between different signal-transduction pathways, may determine the net effect of PDGF.
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PMID:Platelet-derived growth factor. Distinct signal transduction pathways associated with migration versus proliferation. 748 87

PK40erk2 is a MAP kinase which phosphorylates recombinant hTau40 up to 14 moles of phosphate/mole, markedly slowing its electrophoretic mobility. PK40erk2 acting on TAU is expected to cause the appearance of Alzheimer's disease-specific phosphoepitopes, detectable by specific antibodies. Maximal phosphorylation in vitro of hTau40 by PKAcat incorporates only 2-3 moles of phosphate/mole. Consequent, but smaller, reduction in electrophoretic mobility is seen, but not the formation of Alzheimer-specific or hyperphosphorylation-specific epitopes. Phosphorylation of hTau40 by PKAcat sharply reduces the number of phosphates that can now be introduced by PK40erk2 to 5-6 moles/mole, instead of the expected 11 moles/mole. Thus, prior phosphorylation by PKA, a non-proline-directed protein kinase, regulates the conformation of the protein substrate Tau so as to make some sites very much less accessible to phosphorylation by the proline-directed kinase, PK40erk2.
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PMID:Hyperphosphorylation of the cytoskeletal protein Tau by the MAP-kinase PK40erk2: regulation by prior phosphorylation with cAMP-dependent protein kinase A. 748 31


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