Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.
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PMID:Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages. 955 2

Hydrogen peroxide (H2O2) is a potent stimulator of signal-responsive phospholipase A2 (PLA2) in vascular smooth muscle and cultured endothelial cells. We investigated whether H2O2 plays a similar regulatory role in neurons. H2O2 did not stimulate a release of arachidonic acid from cultured neurons when applied alone but strongly enhanced the liberation of arachidonic acid evoked by maximally effective concentrations of either glutamate, the glutamate receptor agonist N-methyl-D-aspartate (NMDA), the muscarinic receptor agonist carbachol, the Na+-channel opener veratridine, or the Ca2+-ionophore ionomycin. The potentiating effects of H2O2 were strongly inhibited in the presence of the PLA2 inhibitor mepacrine, suggesting that the site of action was within the signal responsive arachidonic acid cascade. The enhancing effect of H2O2 was not reversed by protein kinase C inhibitors (chelerythrine chloride or GF 109203X) nor was it mimicked by phorbol ester treatment. H2O2 alone strongly enhanced the levels of immunodetectable activated mitogen-activated protein kinase (activated MAP kinases ERK1 and ERK2) in a Ca2+-dependent manner and this effect was additive with increases in the levels of activated MAP kinase evoked by glutamate. The enhanced release of arachidonic acid, however, was not clearly reversed by the MAP kinase kinase (MEK) inhibitor PD 98059, although this treatment effectively abolished H2O2 activation of MAP kinase. Thus, MAP kinase activation and Ca2+-dependent arachidonic acid release are regulated by oxidative stress in cultured striatal neurons.
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PMID:Hydrogen peroxide enhances signal-responsive arachidonic acid release from neurons: role of mitogen-activated protein kinase. 957 94

Human mesangial cells (HMC) grown in high glucose environments synthesize excessive amounts of extracellular matrix proteins (ECM). The promoter regions of certain ECM genes contain TPA (phorbol ester)-responsive element (TRE) motifs that bind the transcription factor, activator protein-1 (AP-1), a complex of Jun and other phosphoproteins. AP-1 binding to the TRE promoter is regulated by the quantity, composition and post-translational modifications of proteins in the AP-1 complex. We report an increased binding of AP-1 to TRE oligonucleotides in HMC cultured chronically (5 days) in high glucose environments (30 mM d-glucose). This increased binding is not due to differences in the nuclear quantity of AP-1 proteins or in the composition of the AP-1 complex when compared to AP-1 proteins from cells grown in normal glucose (5 mM d-glucose). A 30 mM l-glucose environment also increased AP-1 binding, but to a degree less than d-glucose. The increased AP-1 binding was partly reversed by treatment of HMC with Calphostin C or Bisindolylmaleimide I suggesting a partial role of the protein kinase C (PKC) pathway in mediating AP-1 binding. AP-1 binding was unaffected by treatment of cells with the MEK inhibitor PD 98059. In addition, increased AP-1 binding persisted for at least 48 hours after media glucose concentrations were normalized. The level of Jun-NH2-terminal kinase (JNK) activity and the phosphorylation of the JNK kinase, SEK1, were unchanged by chronic high glucose concentrations. These studies suggest that in HMC cultured in chronic high glucose, post-translational modifications increase the binding of AP-1 to the TRE motif.
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PMID:DNA binding of activator protein-1 is increased in human mesangial cells cultured in high glucose concentrations. 957 31

The effect of stress on the production of cytokine-induced neutrophil chemoattractant (CINC) was examined in rat C6 glioma cells. We studied the production of CINC, an interleukin-8 (IL-8) family protein, with bacterial endotoxin, H2O2, and tumor necrosis factor-alpha (TNF-alpha). Each stress induced CINC mRNA in a concentration-dependent manner. Since stress activates the protein kinases regulating nuclear transcription factors, we examined the effects of protein kinase inhibitors and the over-expression of dominant-negative Ras on CINC mRNA expression. Neither over-expression of dominant-negative Ras nor pretreatment with PD98059 (MEK-1 inhibitor), SB203580 (p38MAPK inhibitor), or GF109203X (protein kinase C (PKC) inhibitor) altered stress-induced CINC mRNA expression. This suggests that the Ras-MAPK, p38MAPK, and PKC pathways are not involved in CINC mRNA expression in glial cells. On the other hand, pretreatment with herbimycin A, a potent tyrosine kinase inhibitor, or Ro31-8220, a non-selective serine/threonine kinase inhibitor, suppressed stress-induced CINC mRNA expression. This indicates that stress-induced CINC mRNA expression is mediated by herbimycin A-, or Ro31-8220-sensitive kinases in glial cells. Since stress activates NF-kappaB and NF-IL6, we examined that the effect of herbimycin A, which suppresses CINC mRNA expression, on NF-kappaB and NF-IL6 activation. Herbimycin A suppressed NF-kappaB but not NF-IL6. These results suggest that in rat glial cells, the factors that induce CINC mRNA expression are mediated by herbimycin A-sensitive NF-kappaB activation, but not through the PKC, Ras-MAPK or p38 MAPK pathways.
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PMID:Induction of cytokine-induced neutrophil chemoattractant in response to various stresses in rat C6 glioma cells. 959 44

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes, i.e. those for c-Fos, skeletal alpha-actin, atrial natriuretic factor, and the alpha3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.
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PMID:Multiple signal transduction pathways link Na+/K+-ATPase to growth-related genes in cardiac myocytes. The roles of Ras and mitogen-activated protein kinases. 961 40

Murine embryonic palate mesenchyme (MEPM) cells are responsive to a number of endogenous factors found in the local embryonic tissue environment. Recently, it was shown that activation of the cyclic AMP (cAMP) or the transforming growth factor beta (TGFbeta) signal transduction pathways modulates the proliferative response of MEPM cells to epidermal growth factor (EGF). Since the mitogen-activated protein kinase (MAPK) cascade is a signal transduction pathway that mediates cellular responsiveness to EGF, we examined the possibility that several signaling pathways which abrogate EGF-stimulated proliferation do so via the p42/p44 MAPK signaling pathway. We demonstrate that EGF stimulates MAPK phosphorylation and activity in MEPM cells maximally at 5 minutes. Tyrosine phosphorylation and activation of MAPK was unaffected by treatment of MEPM cells with TGFbeta or cholera toxin. Similarly, TGFbeta altered neither EGF-induced MAPK tyrosine phosphorylation nor activity. However, the calcium ionophore, A23187, significantly increased MAPK phosphorylation which was further increased in the presence of EGF, although calcium mobilization reduced EGF-induced proliferation. Despite the increase in phosphorylation, we could not demonstrate induction of MAPK activity by A23187. Like EGF, phorbol ester, under conditions which activate PKC isozymes in MEPM cells, increased MAPK phosphorylation and activity but was also growth inhibitory to MEPM cells. The MEK inhibitor, PD098059, only partially abrogated EGF-induced phosphorylation. Likewise, depletion of PKC isozymes partially abrogated EGF-induced MAPK phosphorylation. Inhibition of both MEK and PKC isozymes resulted in a marked decrease in MAPK activity, confirming that EGF uses multiple pathways to stimulate MAPK activity. These data indicate that the MAPK cascade does not mediate signal transduction of several agents that inhibit growth in MEPM cells, and that there is a dissociation of the proliferative response and MAP kinase activation. Furthermore, other signaling pathways known to play significant roles in differentiation of palatal tissue converge with the MAPK cascade and may use this pathway in the regulation of alternative cellular processes.
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PMID:Selective modulation of MAP kinase in embryonic palate cells. 964 14

By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).
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PMID:Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity. 964 41

Different doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.1, 1, 5, and 10 micrograms/kg body wt) were administered i.p. to 21-day-old male Sprague-Dawley rats. Control animals received the same volume of the vehicle (acetone:corn oil, 1:19). Body weight and daily food intake were recorded during the 90-day time course of the study. Random samples of five rats were sacrificed at 34, 49, 62, and 90 days of age. Epidermal growth factor receptor (EGFR) in whole testis was measured, as were the activities of c-Src kinase, protein tyrosine kinase (PTK), mitogen-activated protein 2 kinase (MAP2K also termed as Erk2), protein kinase A (PKA), and protein kinase C (PKC). Testicular tissue from 90-day-old rats was evaluated for histopathology, and sperm numbers in whole testis were counted to estimate daily sperm production. The motility of sperm in the vas deferens and caudal segments of the epididymis of 90-day-old rats was measured by computer assisted sperm analysis (CASA) and the function of the sperm was tested by assessment of acrosome reactions. A dose of 10 micrograms/kg resulted in testicular atrophy and histopathologic examination revealed a decrease in the diameter of the seminiferous tubules. Sertoli cell nuclei were clearly seen, but the spermatogonial population was totally absent. Lower doses of TCDD did not affect testicular histology, but doses as low as 1 microgram/kg significantly decreased testicular sperm numbers and affected some sperm functions (motility parameters and acrosome reactions) in 90-day-old rats. Significant decreases in EGFR were found in 34-day-old rats and this effect on EGFR was sustained until the end of the experiment (90 days). Although TCDD significantly increased c-Src kinase activity in immature and mature rats, opposite effects of TCDD on activities of PTK, PKA, and PKC were found in 34-day-old rats vs 49-, 62-, and 90-day-old rats. When 10 micrograms TCDD/kg was administered to 21-day-old rat, 24-h after c-Src kinase inhibitor geldanamycin, there was no testicular atrophy and no change in the daily sperm production was found. These findings provide evidence for involvement of Src kinase signaling and EGFR in the mechanism by which TCDD disrupts testicular development and subsequently affects testis function.
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PMID:Treatment of rats during pubertal development with 2,3,7,8-tetrachlorodibenzo-p-dioxin alters both signaling kinase activities and epidermal growth factor receptor binding in the testis and the motility and acrosomal reaction of sperm. 965 74

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.
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PMID:Identification of a novel inhibitor of mitogen-activated protein kinase kinase. 966 Aug 36

Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.
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PMID:Platelet-derived growth factor-BB, insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration. 968 41


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