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:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When quiescent dog thyroid epithelial cells in primary culture are stimulated for 48 h with thyrotropin (TSH), forskolin acting through cAMP, or with cAMP-independent mitogens including epidermal growth factor (EGF), hepatocyte growth factor (HGF), and a tumor promoting phorbol ester (TPA), only 30-60% of cells progress through the cell cycle. A more general growth response requires the combination of EGF and TSH or forskolin. In this study we ask whether this intercellular heterogeneity in mitogen sensitivity could depend on a similar heterogeneity at early stages of the mitogenic stimulation process, i.e., at the levels of p42/p44 MAP kinase nuclear translocation and c-Fos protein appearance. We used indirect immunofluorescence microscopy with photometric quantitation and corroborated data using Western blotting. We analyzed the double staining of c-Fos and p42/p44 MAP kinases, since the nuclear translocation of these MAP kinases has been suggested as a key step for the stimulation of c-fos transcription. (i) EGF and HGF induced c-Fos accumulation and MAP kinase translocation in variable fractions of the cell population that corresponded to their relative potency as mitogens. c-Fos appearance and MAP kinase translocation poorly correlated in individual cells. Many cells accumulated c-Fos without any detectable p42/p44 MAP kinase translocation. The heterogeneity of proliferative responses to EGF could be due to the lack of c-Fos or MAP kinase responsiveness of many cells. (ii) TPA induced c-Fos accumulation and MAP kinase translocation within the whole cell population, which did not explain the heterogeneity of the growth response to this factor and showed that these events are not sufficient to elicit DNA synthesis, (iii) TSH and forskolin induced a weak c-Fos accumulation in only a minority of cells but, as previously shown, no p42/p44 MAP kinase phosphorylation and translocation. An important c-Fos expression was thus dispensable for the strong DNA synthesis stimulation exerted by cAMP-dependent mitogens. (iv) Forskolin potentiated the EGF effect on c-Fos expression but not on p42/p44 MAP kinase phosphorylation and translocation. This reflected the fact that EGF induced c-Fos accumulation in 90% of cells in the presence of forskolin but in 30-50% of cells in its absence. This kind of potentiation, which specifically implies an increase in the fraction of responding cells, is termed "generalization" in the present study.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Intercellular heterogeneity of early mitogenic events: cAMP generalizes the EGF effect on c-Fos protein appearance but not on MAP kinase phosphorylation and nuclear translocation in dog thyroid epithelial cells. 758 41

We studied interactions between the mitogen-activated protein kinase (MAPK) signalling pathway and cAMP-protein kinase (PKA) signaling pathway in regulation of mitogenesis of mesangial cells (MC) determined by [3H]thymidine incorporation, with or without added EGF. Forskolin or dibutyryl cAMP strongly (by 60-70%) inhibited [3H]thymidine incorporation into MC. Cilostamide, lixazinone or cilostazol selective inhibitors of cAMP-phosphodiesterase (PDE) isozyme PDE-III, inhibited mitogenesis to similar extent as forskolin and DBcAMP and activated in situ PKA, but without detectable increase in cAMP levels. Cilostamide and cilostazol were more than three times more effective at inhibiting mesangial mitogenesis than rolipram and denbufylline, inhibitors of isozyme PDE-IV, even though PDE-IV was two times more abundant in MC than was PDE-III. On the other hand, when incubated with forskolin, rolipram-enhanced cAMP accumulation was far greater (10-100x) than with cilostamide. EGF increased MAPK activity (+300%); PDE isozyme inhibitors which suppressed mitogenesis also inhibited MAPK. PDE isozyme inhibitors also suppressed PDGF-stimulated MC proliferation. We conclude that cAMP inhibits the mitogen-dependent MAPK-signaling pathway probably by decreasing the activity of Raf-1 due to PKA-catalyzed phosphorylation. Further, we surmise that minor increase in the cAMP pool metabolized by PDE-III is intimately related to regulation of mesangial proliferation. Thus, PDE isozyme inhibitors have the potential to suppress MC proliferation by a focused effect upon signaling pathways.
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PMID:Inhibitors of cyclic nucleotide phosphodiesterase isozymes type-III and type-IV suppress mitogenesis of rat mesangial cells. 761 11

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.
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PMID:cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. 763 71

Stimulation of aortic smooth muscle cells with platelet-derived growth factor BB homodimer (PDGF-BB) leads to the rapid activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MAPKK). Compounds that increase cAMP and activate protein kinase A (PKA)--prostaglandin E2, isoproterenol, cholera toxin, and forskolin--were found to inhibit the PDGF-BB-induced activation of MAPKK and MAPK. Forskolin, but not the inactive analogue 1,9-dideoxyforskolin, inhibited PDGF-BB-stimulated MAPKK and MAPK activation in a dose-dependent manner. PKA antagonism of MAPK signaling was observed at all doses of PDGF-BB or PDGF-AA. PKA did not inhibit MAPKK and MAPK activity in vitro, and MAPKK and MAPK from extracts of forskolin-treated cells could be activated normally with purified Raf-1 and MAPKK, respectively, suggesting that PKA blocked signaling upstream of MAPKK. Neither PDGF-BB-stimulated tyrosine autophosphorylation of the PDGF receptor beta subunit nor inositol monophosphate accumulation was affected by increased PKA activity, suggesting that PKA inhibits events downstream of the PDGF receptor. This study provides an example of cross talk between two important signaling systems activated by physiological stimuli in smooth muscle cells--namely, the PKA pathway and the growth factor-activated MAPK cascade.
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PMID:Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. 769 89

Activation of the mitogen-activated protein kinase (MAP kinase) isoforms ERK1 and ERK2 was investigated in rat adipocytes. Kinase activities were measured by using myelin basic protein as substrate after the isoforms were resolved by Mono Q chromatography or by immunoprecipitation with specific antibodies. Insulin increased the activity of both isoforms by 3- to 4-fold. The beta-adrenergic agonist isoproterenol was without effect in the absence of insulin but markedly reduced the increases in ERK1 and ERK2 activities produced by the hormone. MAP kinase activation was also attenuated by forskolin and glucagon, which increase intracellular cAMP, and by dibutyryl-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP. Thus, increasing cAMP is associated with decreased activation of MAP kinase by insulin. Forskolin also inhibited activation of MAP kinase by several agents (epidermal growth factor, phorbol 12-myristate 13-acetate, and okadaic acid) that act independently of insulin receptors. Moreover, forskolin did not inhibit insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate IRS-1. Therefore, the inhibitory effect on MAP kinase did not result from compromised functioning of the insulin receptor. The inhibitory effect was not confined to adipocytes, as forskolin and dibutyryl-cAMP inhibited the increase in MAP kinase activity by phorbol 12-myristate 13-acetate in wild-type CHO cells. In contrast, these agents did not inhibit MAP kinase activity in mutant CHO cells (line 10248) that express a cAMP-dependent protein kinase resistant to activation by cAMP. Our results suggest that activation of cAMP-dependent protein kinase represents a general counter-regulatory mechanism for opposing MAP kinase activation.
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PMID:Increasing cAMP attenuates activation of mitogen-activated protein kinase. 769 90

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 microM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation-sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 microM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 microM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 microM okadaic acid treatment; similar results were seen using the calcineurin-selective inhibitor cypermethrin. Treatment of human slices with 10 microM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau-directed kinase, 42-kDa mitogen-activated protein kinase (p42mapk), was activated by okadaic acid (> 100 nM) but not FK-520 (5 microM). Nerve growth factor (100 ng/ml) activated p42mapk, particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 microM) antagonized the effects of nerve growth factor on both p42mapk activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.
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PMID:Tau phosphorylation in brain slices: pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase. 772 35

Effects of cAMP on insulin-stimulated mitogen-activated protein (MAP) kinase pathway were examined using rat hepatoma H4EII cells. MAP kinase was rapidly activated and reached a peak 3 min after the stimulation by insulin. Forskolin (1 microM) and 8(4-chlorophenylthio)cAMP (8-CPT-cAMP) (0.1 mM) inhibited the insulin-stimulated MAP kinase activity. Pretreatment of the cells with H-8 (50 microM), a cAMP-dependent protein kinase inhibitor, enhanced the insulin-stimulated MAP kinase activity and partially restored the inhibitory effect of cAMP. Furthermore, insulin-induced phosphorylation of MAP kinase was inhibited by 8-CPT-cAMP, and the inhibition was restored by H-8. 8-CPT-cAMP did not inhibit the autophosphorylation of insulin receptor. These data indicate that elevation of intracellular cAMP blocks the insulin-stimulated MAP kinase pathway downstream of insulin receptor.
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PMID:cAMP inhibits the insulin-stimulated mitogen-activated protein kinase pathway in rat hepatoma H4EII cells. 804 24

G-protein coupled Angiotensin II receptors (AT1A), mediate cellular responses through multiple signal transduction pathways. In AT1A receptor-transfected CHO-K1 cells (T3CHO/AT1A), angiotensin II (AII) stimulated a dose-dependent EC50 = 3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT1, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or ca2+/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [3H]thymidine incorporation in T3CHOA/AT1A cells. AII (1 microM) significantly inhibited cell number (51% at 96 h) and [3H]thymidine incorporation of 68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT1 receptor. Forskolin (10 microM) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [3H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity an tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT1A cells, in particular at 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.
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PMID:A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells. 860 15

Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5'-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0 +/- 5.4 nM, Bmax = 707 +/- 11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca(2+)-independent/wortmannin-sensitive pathway in CHO cells.
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PMID:Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells. 864 36

In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation.
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PMID:A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells. 883 45


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