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)

CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.
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PMID:Autophosphorylation of the mammalian multifunctional protein that initiates de novo pyrimidine biosynthesis. 1198 31

Reactive oxygen species released during the respiratory burst are known to participate in cell signaling. Here we demonstrate that hydrogen peroxide produced by the respiratory burst activates AP-1 binding. Stimulation of the macrophage cell line NR8383 with respiratory burst agonists ADP and C5a increased AP-1 binding activity. Importantly, this increase in binding was blocked by catalase, confirming mediation by endogenous H(2)O(2). Moreover, exogenously added H(2)O(2) mimicked the agonists, and also activated AP-1. Antibodies revealed that the activated AP-1 complex is composed predominantly of c-Fos/c-Jun heterodimers. Treatment of the cells with ADP, C5a and H(2)O(2) (100 microM) all increased the phosphorylation of c-Jun. c-Fos protein was increased in cells treated with C5a or high dose (200 microM) H(2)O(2), but not in cells treated with ADP. The MEK inhibitor, PD98059, partially blocked the C5a-mediated increase in AP-1 binding. A novel membrane-permeable peptide inhibitor of JNK, JNKi, also inhibited AP-1 activation. Together these data suggest that C5a-mediated AP-1 activation requires both the activation of the ERK and JNK pathways, whereas activation of the JNK pathway is sufficient to increase AP-1 binding with ADP. Thus, AP-1 activation joins the list of pathways for which the respiratory burst signals downstream events in the macrophage.
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PMID:AP-1 activation through endogenous H(2)O(2) generation by alveolar macrophages. 1205 68

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.
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PMID:Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2. 1214 48

In the present study, we addressed the question of a putative relevance of Rho proteins in tumour progression by analysing their expression on protein and mRNA level in breast tumours. We show that the level of RhoA, RhoB, Rac1 and Cdc42 protein is largely enhanced in all tumour samples analysed (n=15) as compared to normal tissues originating from the same individual. The same is true for (32)P-ADP-ribosylation of Rho proteins which is catalysed by Clostridium botulinum exoenzyme C3. Also the amount of Rho-GDI and ERK2 as well as the level of overall (32)P-GTP binding activity was tumour-specific elevated, yet to a lower extent than Rho proteins. Although the amount of Rho proteins was enhanced in tumours, most of them did not show changes in rho mRNA expression as compared to the corresponding normal tissue. Thus, elevated gene expression seems not to be the underlying mechanism of tumour-specific overexpression of Rho proteins. Sequence analysis of RhoA, RhoB, RhoC and Rac1 failed to detect any mutations in both the GTP-binding site and effector binding region. By analysing >50 tumour samples, the amount of RhoA-like proteins (i.e. RhoA, B, C), but not of Rac1, was found to significantly increase with histological grade and proliferation index. Rho protein expression was neither related to p53 nor to HER-2/neu oncogene status. Expression of rho mRNAs did not show a significant increase with histological grade. Overall the data show that (1) Rho proteins are overexpressed in breast tumours (2) overexpression is not regulated on the mRNA level (3) the expression level of RhoA-like proteins correlates with malignancy and (4) Rho proteins are not altered by mutation in breast tumours.
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PMID:Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters. 1223 74

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.
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PMID:Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor. 1242 66

Homocysteine (HC) is a neurotoxic amino acid that accumulates in several neurological disorders including Alzheimer's disease (AD). We examined the consequences of treatment of cultured murine cortical neurons with HC. Homocysteine-induced increases in cytosolic calcium, reactive oxygen species, phospho-tau immunoreactivity and externalized phosphatidyl serine (indicative of apoptosis). Homocysteine-induced calcium influx through NMDA channel activation, which stimulated glutamate excitotoxicity, as evidenced by treatment with antagonists of the NMDA channel and metabotropic glutamate receptors, respectively. The NMDA channel antagonist MK-801 reduced tau phosphorylation but not apoptosis after HC treatment, suggesting that HC-mediated apoptosis was not due to calcium influx. Apoptosis after HC treatment was reduced by co-treatment with 3-aminobenazmidine (3ab), an inhibitor of poly-ADP-ribosome polymerase (PARP), consistent with previous reports that ATP depletion by PARP-mediated repair of DNA strand breakage mediated HC-induced apoptosis. Treatment with 3ab did not reduce tau phosphorylation, however, therefore hyperphosphorylation of tau may not contribute to HC-induced apoptosis under these conditions. Inhibition of mitogen-activated protein kinase by co-treatment with the kinase inhibitor PD98059 inhibited tau phosphorylation but not apoptosis after HC treatment. HC accumulation reduces cellular levels of S-adenosyl methionine (SAM); co-treatment with SAM reduced apoptosis, suggesting that inhibition of critical methylation reactions may mediate HC-induced apoptosis. These findings indicate that HC compromises neuronal homeostasis by multiple, divergent routes.
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PMID:Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. 1242 37

ADP-ribosylation has been coupled to intracellular events associated with smooth muscle cell vasoreactivity, cytoskeletal integrity and free radical damage. Additionally, there is evidence that ADP-ribosylation is required for smooth muscle cell proliferation. Our investigation employed selective inhibitors to establish that mono-ADP-ribosylation and not poly(ADP-ribosyl)ation was necessary for the stimulation of DNA synthesis by mitogens. Mitogen treatment increased concomitantly the activity of both soluble and particulate mono-ADP-ribosyltransferase, as well as the number of modified proteins. Inclusion of meta-iodobenzylguanidine (MIBG), a selective decoy substrate of arginine-dependent mono-ADP-ribosylation, prevented the modification of these proteins. MIBG also blocked the stimulation of DNA and RNA synthesis, prevented smooth muscle cell migration and suppressed the induction of c-fos and c-myc gene expression. An examination of relevant signal transduction pathways showed that MIBG did not interfere with MAP kinase and phosphatidylinositol 3-kinase stimulation; however, it did inhibit phosphorylation of the Rho effector, PRK1/2. This novel observation suggests that mono-ADP-ribosylation participates in a Rho- dependent signalling pathway that is required for immediate early gene expression.
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PMID:Endogenous mono-ADP-ribosylation mediates smooth muscle cell proliferation and migration via protein kinase N-dependent induction of c-fos expression. 1249 80

Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, alphaIIbbeta3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to alphaIIbbeta3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the alphaIIbbeta3 with mutated beta3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type alphaIIbbeta3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of alphaIIbbeta3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to alphaIIbbeta3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified alphaIIbbeta3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with alphaIIbbeta3 and can change alphaIIbbeta3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin.
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PMID:Thrombospondin-bound integrin-associated protein (CD47) physically and functionally modifies integrin alphaIIbbeta3 by its extracellular domain. 1273 72

The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 micromol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38(MAPK), and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase activation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms.
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PMID:Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms. 1280 10

Current evidence suggests that amyloid beta peptides (Abeta) may play a major role in the pathogenesis of Alzheimer's disease by eliciting oxidative stress and neuronal apoptosis. In this study we have used differentiated SK-N-BE neurons to investigate molecular mechanisms and regulatory pathways underlying apoptotic neuronal cell death elicited by Abeta(1-40) and Abeta(1-42) peptides as well as the relationships between apoptosis and oxidative stress. Abeta peptides, used at concentrations able to induce oxidative stress, elicit a classic type of neuronal apoptosis involving mitochondrial regulatory proteins and pathways (i.e. affecting Bax and Bcl-2 protein levels as well as release of cytochrome c in the cytosol), poly-ADP rybose polymerase cleavage and activation of caspase 3. This pattern of neuronal apoptosis, that is significantly prevented by alpha-tocopherol and N-acetylcysteine and completely abolished by specific inhibitors of stress-activated protein kinases (SAPK) such as JNKs and p38(MAPK), involved early elevation of p53 protein levels. Pretreatment of neurons with alpha-pifithrin, a specific p53 inhibitor, resulted in a 50-60% prevention of Abeta induced apoptosis. These results suggest that oxidative stress - mediated neuronal apoptosis induced by amyloid beta operates by eliciting a SAPK-dependent multiple regulation of pro-apoptotic mitochondrial pathways involving both p53 and bcl-2.
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PMID:Multiple signaling events in amyloid beta-induced, oxidative stress-dependent neuronal apoptosis. 1282 55


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