Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two naturally occurring mutant insulin receptors, Arg-1174 --> Gln and Leu-1178 --> Pro, found in patients with dominantly inherited Type A insulin resistance, showed unusual signaling properties when stably expressed in Chinese hamster ovary (CHO) cells. Both mutant receptors were expressed on the cell surface and bound insulin normally, but showed markedly impaired autophosphorylation in response to insulin. In addition, the in vitro tyrosine kinase activity of both mutant receptors toward an artificial substrate was also severely impaired. Despite these defects of kinase activity, anti-phosphotyrosine immunoblotting of whole cell lysates and anti-phosphotyrosine immunoprecipitation of 32P-labeled cells showed insulin-stimulated tyrosine phosphorylation of a protein of approximately 185 kDa to an extent comparable to that seen in CHO cells expressing wild-type human insulin receptors. Anti-insulin receptor substrate-1 (IRS-1) immunoprecipitation followed by anti-phosphotyrosine immunoblotting confirmed that this tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells expressing an insulin receptor mutated at the ATP binding site (Lys-1030 --> Arg) showed no insulin-stimulated autophosphorylation or phosphorylation of IRS-1. Despite exhibiting apparently normal insulin stimulation of IRS-1 tyrosine-phosphorylation, cells expressing the Arg-1174 --> Gln or Pro-1178 --> Leu receptors showed marked impairment in insulin stimulation of glycogen synthesis, thymidine incorporation, and activation of MAP kinase. The inability of these mutant receptors to signal normally to metabolic and mitogenic responses suggests that insulin-stimulated tyrosine phosphorylation of IRS-1 alone is insufficient to fully mediate insulin action.
 ...
PMID:Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin. Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action. 863 49

Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.
 ...
PMID:Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5'-triphosphate. 863 22

The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.
 ...
PMID:Substrate specificity of IphP, a cyanobacterial dual-specificity protein phosphatase with MAP kinase phosphatase activity. 865 37

In an effort to determine the role of protein kinase C-delta (PKC-delta) in cellular transformation mediated by the sis proto-oncogene, we cotransfected expression vectors containing cDNAs that encode for c-sis with an ATP binding mutant of PKC-delta (PKC-delta K376R) or wild type PKC-delta (PKC-delta WT) into NIH3T3 cells. Our results showed that expression of PKC-delta K376R severely impaired Sis-induced focus formation, whereas cotransfection of PKC-delta WT cDNA had no effect on Sis-mediated transformation. Consistent with this result, PKC-delta K376R expression also inhibited PDGF-BB-mediated anchorage-independent colony formation. While cotransfection of a vector containing a dominant negative mutant of ras (N17 ras) cDNA potently inhibited Sis-induced transformation, the expression of PKC-delta K376R did not block transformation mediated by v-H-Ras or v-Raf. In addition, PDGF-BB-induced Raf and mitogen-activated protein kinase activation, which are known to be downstream molecules in the Ras cascade, were not affected by the expression of PKC-delta K376R, indicating that PKC-delta and Ras are segregated in mediating Sis-induced transformation. Interestingly, expression of PKC-delta K376R strongly reduced TPA responsive element (TRE) transactivation induced by PDGF stimulation, suggesting that activation of TRE-containing genes, which may be involved in Sis-mediated transformation, are negatively regulated by expression of PKC-delta K376R.
 ...
PMID:Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells. 876 Dec 94

The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha). The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin. While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases. The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.
 ...
PMID:Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists. 876 79

To summarize the regulation of cPLA2, we have proposed a model for the activation of cPLA2 based both on our previous studies (Clark et al., 1991; Lin et al., 1993) and the work of many others (Fig. 5). In this model, cPLA2 is tightly regulated by multiple pathways, including those that control Ca2+ concentration, phosphorylation states and cPLA2 protein levels, to exert both rapid and prolonged effects on cellular processes, such as inflammation. cPLA2 is rapidly activated by increased intracellular Ca2+ concentration and phosphorylation by MAP kinase. When cells are stimulated with a ligand for a receptor, such as ATP or PDGF, PLC is activated via either a G protein-dependent or -independent process, leading to the production of diacylglycerol (DAG) and inositol triphosphate (IP3). The rise in these intracellular messengers cause the activation of PKC and mobilization of intracellular Ca2+. Alternatively, the increase in intracellular Ca2+ can result from a Ca2+ influx. Increased Ca2+ acts through the CaLB domain to cause translocation of cPLA2 from the cytosol to the membrane where its substrate, phospholipid, is localized. This step is essential for the activation of cPLA2 and may account for the partial activation of cPLA2 in the absence of phosphorylation. MAP kinase activation can occur through both PKC-dependent and -independent mechanisms (Cobb et al., 1991; Posada and Cooper, 1992; Qiu and Leslie, 1994). In many cases, this pathway is also G protein-dependent. Activated MAP kinase phosphorylates cPLA2 at Ser-505, causing increased enzymatic activity of cPLA2, which is realized only upon translocation of cPLA2 to the membrane. Therefore, full activation of cPLA2 requires both increased cytosolic Ca2+ and cPLA2 phosphorylation at Ser-505. In a more delayed response, cPLA2 activity in the cells can be controlled by changes in its expression levels, such as in response to inflammatory cytokines and certain growth factors. Thus the expression level of cPLA2 is regulated by both transcriptional and post-transcriptional mechanisms.
 ...
PMID:Cytosolic phospholipase A2. 877 86

The mitogenic effect of extracellular ATP was examined in cultured rat aortic smooth muscle cells (VSMCs). ATP, 2-methylthio-ATP, and ADP stimulated [3H]thymidine and [3H]leucine incorporation and cell growth. AMP, adenosine, UTP, and P2x agonists showed little of these effects. Reactive blue 2, a P2Y purinoceptor antagonist, was effective in suppressing the mitogenic effect of ATP and 2-methylthio-ATP, indicating that extracellular ATP-induced VSMC proliferation is mediated by P2Y purinoceptors. The P2Y purinoceptor activation was coupled to a pertussis toxin (PTX)-insensitive G protein (Gq) and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C (PKC), Raf-1, and mitogen-activated protein kinase (MAPK) in VSMCs. In response to ATP, both 42-and 44-kDa MAPKs were activated, and tyrosine was phosphorylated. Western blot analysis using PKC isozyme-specific antibodies indicated that VSMCs express PKC-alpha, PKC-delta, and PKC-zeta. A complete down-regulation of PKC-alpha and PKC-delta was seen after 24-hr treatment with 12-O-tetradecanoylphorbol-13-acetate. When cells were pretreated with 12-O-tetradecanoyl-phorbol-13-acetate for 24 hr and subsequently challenged with ATP, Raf-1 activation and 42-kDa as well as 44-kDa MAPK tyrosine phosphorylation failed to be induced. These results demonstrate that ATP-induced Raf-1 and MAPK activations involve the activation of PKC-alpha and PKC-delta. P2Y purinoceptor stimulation with ATP also caused accumulation of c-fos and c-myc mRNAs. Both Reactive blue 2 and staurosporine significantly blocked this increase by ATP. In conclusion, the mitogenic effect of ATP seemed to be triggered by activation of the Gq protein-coupled P2Y purinoceptor that led to the formation of inositol trisphosphate and activation of PKC. PKC and, in turn, Raf-1 and MAPK were then activated, leading eventually to DNA synthesis and cell proliferation.
 ...
PMID:Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells. 949 67

1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor.
 ...
PMID:Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells. 888 34

Extracellular ATP is known to activate intracellular enzymes in astrocytes via P2 purinoceptors that appear to play important physiological and pathological roles in these supporting brain cells. In this study, major P2 purinoceptor subtypes on astrocytes of neonatal rat cerebral cortices were identified in receptor expression experiments, when astrocytic messenger RNA was injected into Xenopus oocytes and recombinant P2 purinoceptors were characterized pharmacologically. In messenger RNA-injected oocytes, ATP evoked inward chloride currents (ICl,Ca) typical of stimulating metabotropic receptors that release intracellular Ca2+. Half-maximal activation with ATP occurred at 40 nM: the Hill coefficient was 0.5, which indicated that ATP stimulated two subtypes of P2 purinoceptor. UTP and 2-methylthioATP were the most active (and equipotent) of a series of nucleotides activating recombinant P2 purinoceptors. These results indicated that the two P2 purinoceptors expressed by astrocytic messenger RNA were of P2U and P2Y subtypes. Responses to ATP were antagonized by the P2 purinoceptor antagonist (suramin) but not by the P1 purinoceptor blocker (sulphophenyltheophylline). Findings in expression studies were confirmed in assays of intracellular signalling systems using primary cultures of rat astrocytes. UTP and 2-methylthioATP stimulated mitogen-activated protein kinase to the same extent as ATP, although UTP was less potent than either ATP or 2-methylthioATP. Both UTP and ATP increased intracellular Ca2+ (as measured by fura-2/AM luminescence) which, in cross-desensitization experiments, indicated the involvement of two subtypes of P2 purinoceptors. In conclusion, rat cortical astrocytes express two major subtypes (P2U and P2Y) of metabotropic ATP receptor which, when activated, raise intracellular Ca2+ and also stimulate mitogen-activated protein kinase.
 ...
PMID:P2 purinoceptors in rat cortical astrocytes: expression, calcium-imaging and signalling studies. 889 85

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y2 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown that both the endothelial P2Y purinoceptors are linked to activation of MAPK, and that activation of this pathway is a requirement for the stimulation by ATP/ADP of endothelial PGI2 production.
 ...
PMID:Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production. 894 91


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>