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)

The activation of human mitogen-activated protein kinase kinase 1 (MKK1) is achieved by phosphorylation at Ser218 and Ser222 within a regulatory loop. Partial activation was achieved by replacing these residues with aspartic/glutamic acid. Higher activity was obtained by introducing four acidic residue substitutions in the regulatory loop, indicating that acidic residues in the loop stabilize an active configuration by the introduction of negative charge. Activation of MKK1 is also achieved by deleting residues 44-51, N-terminal to the consensus catalytic core. Although substitution of residues within this segment by alanine does not affect activity, introduction of proline residues elevates kinase activity, indicating that activation results from perturbation of secondary structure within residues 44-51. Pseudosubstrate inhibition, a commonly observed mechanism of kinase regulation, is not operative in this process. Both the acidic substitutions and the N-terminal deletion increase Vmax, V/K(m),ERK2, and V/K(m),ATP, as is also observed following phosphorylation of wild-type MKK1. A synergistic enhancement of these steady-state rate parameters occurs upon combining the mutations, suggesting that conformational changes induced by mutagenesis together mimic those seen upon phosphorylation.
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PMID:Interdependent domains controlling the enzymatic activity of mitogen-activated protein kinase kinase 1. 895 7

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.
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PMID:Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. 903 87

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.
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PMID:Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways. 904 86

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/1 culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [gamma-32P]ATP and used to probe protein-protein or protein-nucleic acid interactions.
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PMID:A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins. 904 44

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.
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PMID:Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes. 909 73

The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.
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PMID:A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. 909

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2- at the expense of NADPH: 2 O2 + NADPH --> 2 O2- + NADP+ + H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47(PHOX), an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47(PHOX). The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47(PHOX) is more physiological than activation by amphiphiles, because the mutant p47(PHOX) S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.
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PMID:Kinase-dependent activation of the leukocyte NADPH oxidase in a cell-free system. Phosphorylation of membranes and p47(PHOX) during oxidase activation. 911 Sep 96

The structure of mitogen-activated protein (MAP) kinase p38 has been solved at 2.1-A to an R factor of 21.0%, making p38 the second low activity MAP kinase solved to date. Although p38 is topologically similar to the MAP kinase ERK2, the phosphorylation Lip (a regulatory loop near the active site) adopts a different fold in p38. The peptide substrate binding site and the ATP binding site are also different from those of ERK2. The results explain why MAP kinases are specific for different activating enzymes, substrates, and inhibitors. A model presented for substrate and activator interactions has implications for the evolution of protein kinase cascades.
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PMID:The structure of mitogen-activated protein kinase p38 at 2.1-A resolution. 912 94

Mitogen-activated protein (MAP) kinase has been implicated in signal transduction pathways that regulate cell cycle progression during the proliferation of eukaryotic cells. Previous studies have shown that a rapid burst of cell proliferation is a major event of the development of mammalian palatal shelves in a vertical direction. The present study analyzed the involvement of MAP kinase during the vertical development of the secondary palate in hamster. Palates were dissected at various times between days 10:00 and 12:00 of gestation, homogenized, centrifuged and fractionated on a Mono Q column by fast protein liquid chromatography. The fractions were assayed for phosphotransferase activity toward myelin basic protein, and also toward a synthetic peptide APRTPGGRR (S5), which was more specifically utilized by MAP kinase. The data showed that MAP kinase activity increased during the initial phase, i.e., between days 10:00 and 11:12, and then decreased during the latter half of vertical palate development, i.e., between days 11:12, and 12:00 of gestation. Western blotting studies, using antibodies raised against the subdomain I ATP binding sequence (GEGA), subdomain III (ERK1-III), and the C-terminus (ERK1-CT) of MAP kinases, demonstrated the presence of both the 42-kDa and 44-kDa MAP kinase isoforms between days 10:12 and 12:12 of gestation. A monoclonal antibody (4G10), which detects phosphotyrosine, demonstrated phosphorylation of both the 42-kDa and 44-kDa isoforms. The amount of protein remained constant during vertical palatal shelf development indicating that the differential activity of MAP kinase was most likely due to post-translational modification (i.e., phosphorylation). There was a good correlation between the temporal expression of MAP kinase activity and the rates of cell proliferation in the developing vertical palate suggesting a possible involvement of MAP kinase in regulation of cell proliferation during secondary palate development.
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PMID:The activation of MAP kinase during vertical palatal shelf development in hamster. 912 68

1. The expression and functional roles of protein kinase C (PKC) isoenzymes in purinoceptor signalling have been examined in rat renal mesangial cells. 2. It is observed that rat mesangial cells express four PKC isoenzymes, PKC-alpha, -delta, -epsilon and zeta, as determined by Western blot analysis. No PKC-beta, -gamma, -eta, -theta, or -mu isoforms were detected. 3. By using specific PKC inhibitors and down-regulation experiments we provide evidence that PKC alpha acts as a negative feedback regulator of ATP- and UTP-stimulated phosphoinositide turnover, whereas PKC epsilon triggers arachidonic acid release and subsequent prostaglandin synthesis and stimulates a phosphatidylcholine-hydrolysing phospholipase D. Moreover, PKC delta may activate the mitogen-activated protein kinase cascade and thus promote mesangial cell proliferation in response to extracellular ATP and UTP. 4. In summary our data identify mesangial cells in culture as an excellently characterized cell culture system with well-defined functions of PKC isoenzymes. Functional identification of PKC isoenzymes involved in specific cell responses is one of the most promising steps towards understanding of molecular mechanisms of cell regulation and identifies new targets for drug development.
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PMID:Regulatory functions of protein kinase C isoenzymes in purinoceptor signalling in mesangial cells. 913 6


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