Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth-arrested WS-1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+ exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA-induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H-7 (100 microM). No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 microM) or the intracellular calcium-chelator BAPTA. In contrast, the thrombin-induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 microM H-7. The thrombin-induced activation of Na+/H+ exchange was inhibited by 200 microM TMB8 or pretreatment with BAPTA. PMA caused translocation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth-arrested WS-1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]-thymidine incorporation in cells serum-deprived for 48 hours. In addition, PMA inhibited thrombin-induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin-induced pathway correlates with a mitogenic response.
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PMID:WS-1 human fibroblasts contain distinct calcium and protein kinase C-mediated pathways for activation of Na+/H+ exchange: contrasting effects of thrombin and PMA. 199 77

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.
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PMID:Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C. 205 Jun 83

Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 microM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 micrograms/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis. Northern blot analysis indicated that thrombin induced an increase in the mRNA levels of t-PA and of PAI-1. We conclude that thrombin increases DNA synthesis in human mesangial cells and enhances the synthesis of both t-PA and PAI-1. The latter is released in a large excess as compared to t-PA. Hence, thrombin may have a role in provoking a localized hypofibrinolytic state and may contribute to the persistence of glomerular fibrin deposits during proliferative glomerulonephritis.
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PMID:Thrombin regulates components of the fibrinolytic system in human mesangial cells. 212 90

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositol-containing phospholipids relied upon the separation of the products [3H]inositol mono-, bis- and tris-phosphates by Dowex-1 chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone H1 in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95% of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 microgram/ml) did not enhance phosphoinositide production, and at higher concentrations (50 micrograms/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.
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PMID:Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets. 216 Feb 34

The stimulation of human platelets by thrombin leads to the activation of phospholipases C and A2, protein kinases, formation of 3-inositol phospholipids and mobilization of Ca2+. These biochemical reactions closely parallel platelet shape change, granular secretion and aggregation. The membrane-bound transducers for the thrombin receptor seem to be the heterotrimeric G protein Gi2 and the ras-related G protein rap 1-b. Phosphorylation of rap 1-b by the action of the cyclic AMP-dependent protein kinase seems to uncouple the thrombin receptor from phospholipases. This causes inhibition of the formation of second messenger molecules and the onset of physiological responses.
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PMID:The signal transduction induced by thrombin in human platelets. 216 95

Octimibate inhibited ADP- and collagen-induced platelet aggregation in human, rabbit and rat platelet-rich plasma. Washed human platelets treated with octimibate had elevated cyclic AMP (cAMP) levels and cAMP-dependent protein kinase activity. When whole platelets were incubated with radiolabeled phosphate, octimibate produced an increase in the phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. This pattern of protein phosphorylation is identical to that observed when the platelets were treated with forskolin, phosphodiesterase inhibitors or other compounds that elevate platelet cAMP levels. Octimibate also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets, which is consistent with octimibate's ability to elevate platelet cAMP levels. When isolated platelet plasma membranes were treated with octimibate, adenylate cyclase activity was stimulated, reaching maximal activation at 1 microM octimibate. (The maximal activation of adenylate cyclase observed with octimibate is 70-75% of that observed with 10 microM PGE1.) This stimulation of platelet adenylate cyclase activity was enhanced by GTP. Octimibate competed for radiolabeled prostaglandin E1 and lloprost binding to isolated platelet membranes at submicromolar concentrations, but did not compete with radiolabeled prostaglandin D2 binding. These studies suggest that octimibate inhibits platelet aggregation by activating platelet adenylate cyclase through stimulation of platelet prostacyclin receptors.
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PMID:Octimibate inhibition of platelet aggregation: stimulation of adenylate cyclase through prostacyclin receptor activation. 217 92

alpha-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by alpha-thrombin was clearly distinct from the alpha subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively Gi alpha and Gs alpha; the 47-kDa protein that is phophorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.
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PMID:Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets. 233 84

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.
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PMID:Role of cyclic-GMP in relaxations of vascular smooth muscle. 240 83

1. The reduction of cytoplasmic free calcium, [Ca2+]i following stimulation, has been investigated in fura-2-loaded human platelets in the presence of low extracellular calcium concentration. Thrombin produced a rapid rise in [Ca2+]i which then fell back to the basal level within 2 min. 2. Ionomycin produced a rapid elevation in [Ca2+]i which then declined to a plateau well above the basal calcium level. The addition of thrombin after ionomycin accelerated the decline in [Ca2+]i back towards basal levels, an action mimicked by phorbol myristate acetate (PMA). 3. Thrombin promoted the efflux of 45Ca2+ from cells co-loaded with fura-2 and the isotope. Ionomycin also promoted an efflux of 45Ca2+ which was increased by the subsequent addition of thrombin or PMA. These results confirm the ability of thrombin and PMA to stimulate Ca2+ removal from the cells. 4. The complete substitution of extracellular Na+ with N-methyl-D-glucamine (NMDG) did not alter the time course of the return of [Ca2+]i to basal following stimulation by thrombin, nor the ability of thrombin or PMA to promote Ca2+ efflux after elevation of [Ca2+]i by ionomycin. 5. The insensitivity to external Na+ suggests that the stimulated Ca2+ efflux is mediated by a Ca2+-ATPase rather than Na+-Ca2+ exchange. This pump does not appear to be activated by Ca2+-calmodulin since [Ca2+]i remains high when elevated by ionomycin. The ability of PMA to stimulate removal suggests that its known target, protein kinase C, can stimulate the Ca2+ pump. Forskolin, which stimulates adenylate cyclase, did not stimulate a fall in [Ca2+]i in the presence of ionomycin, indicating that cyclic AMP-dependent protein kinase does not stimulate Ca2+ extrusion.
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PMID:Stimulated calcium efflux from fura-2-loaded human platelets. 245 43

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.
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PMID:Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin. 246 67


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