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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activity-dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down-regulation of synapse effectiveness. We further demonstrate that a thrombin receptor-activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP-evoked synapse decrement is unaffected by a specific protein kinase A blocker, H-89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity-dependent synapse reduction expressed in our system.
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PMID:The thrombin receptor mediates functional activity-dependent neuromuscular synapse reduction via protein kinase C activation in vitro. 1002 79

Acute and chronic interstitial lung diseases are accompanied by evidence of inflammation and vascular injury. Thrombin activity in bronchoalveolar lavage fluid from such conditions is often increased, as well as interleukin (IL)-8. We observed that conditioned medium from lung fibroblasts exposed to thrombin has chemotactic activity for polymorphonuclear cells, and that this activity can be abolished by antibody to IL-8. We report that thrombin stimulates expression of IL-8 in human lung fibroblasts on both the messenger RNA and protein levels in a time- and dose-dependent manner. Stimulation of IL-8 expression by thrombin is inhibited by specific thrombin inhibitors. Synthetic thrombin receptor agonist peptide-14 mimics thrombin's stimulation of IL-8 expression in a dose-dependent manner consistent with the idea that upregulation of IL-8 by thrombin in human lung fibroblasts requires cleavage of proteolytically activated receptor-I. We demonstrate further that thrombin-induced IL-8 synthesis is regulated by protein kinase (PK) C. PKC-gamma may be involved in the upregulation of lung fibroblast IL-8 by thrombin because stimulation of lung fibroblasts with thrombin caused significant upregulation of PKC-gamma and because PKC-gamma antisense oligonucleotides inhibited the accumulation of PKC-gamma protein and IL-8 protein. Our data suggest that the PKC-gamma isoform increase observed after thrombin stimulation is required for thrombin-induced IL-8 formation by human lung fibroblasts.
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PMID:Thrombin upregulates interleukin-8 in lung fibroblasts via cleavage of proteolytically activated receptor-I and protein kinase C-gamma activation. 1065 45

Stimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PARI (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A1 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.
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PMID:Identification of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of protease-activated receptor 1 (thrombin receptor) in human platelets. 1078 Mar 27

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs) and plays an important role in the progression of atherosclerosis. Although recent reports have suggested that cAMP response element-binding protein (CREB) is necessary for the survival of neuronal cells, the role of CREB in VSMC proliferation is not determined. We examined the role of CREB in thrombin-induced VSMC proliferation and the effect of thrombin on phosphorylation of CREB at Ser133, which is a critical marker for activation by Western blot analysis. Thrombin induced phosphorylation of CREB in a dose-dependent manner. An oligopeptide, SFLLRN, which activates the thrombin receptor, also induced the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase or inhibition of p38 mitogen-activated protein kinase suppressed the thrombin-induced CREB phosphorylation. Inhibition of the epidermal growth factor receptor by AG1478 also inhibited the thrombin-induced CREB phosphorylation. Overexpression of the dominant-negative form of CREB inhibited thrombin-induced c-fos mRNA expression and incorporation of [(3)H]thymidine and [(3)H]leucine. These results suggest that CREB-dependent gene transcription plays a critical role in thrombin-induced proliferation and hypertrophy of VSMCs. Transactivation of the epidermal growth factor receptor and 2 mitogen-activated protein kinase pathways are involved in this process. CREB may be a novel transcription factor mediating the vascular remodeling process induced by thrombin.
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PMID:cAMP response element-binding protein mediates thrombin-induced proliferation of vascular smooth muscle cells. 1170 63

1 The lysophospholipids, lysophosphatidic acid and sphingosine 1-phosphate, have been reported to activate platelets. Here we examined effects of the naturally occurring related sphingosylphosphorylcholine (SPC) on human platelet function. 2 Platelet activation was determined as aggregation, elevation of intracellular Ca(2+) concentrations, surface expression of P-selectin, GP 53, and GP IIb/IIIa neoepitope PAC-1, and of fibrinogen binding to the platelet surface. 3 Platelets were activated by ADP (5 and 20 micro M), the thrombin receptor-activating peptide TRAP-6 (5 and 20 micro M), the thromboxane A(2) mimetic U-46619 (1 micro M) and collagen (20 and 50 micro g ml(-1)) but not by SPC (up to 20 micro M). 4 SPC concentration-dependently (IC(50) approximately 1-10 micro M) inhibited activation of washed human platelets in response to all of the above agonists, with almost complete inhibition occurring at 20 micro M SPC. 5 The SPC stereoisomers, D-erythro SPC and L-threo SPC, exhibited similar concentration-response curves in inhibiting 20 micro M ADP-induced platelet aggregation, suggesting that SPC did not act via specific lysophospholipid receptors. 6 Although SPC slightly activated platelet protein kinase A (as assessed by VASP phosphorylation), this effect could not explain the marked platelet inhibition. Possible protein kinase C inhibition also did not explain the inhibition of platelet activation by SPC. On the other hand, SPC suppressed agonist-induced Ca(2+) mobilization and phospholipase C stimulation. 7 These results indicate that the lysophospholipid SPC is an effective inhibitor of human platelet activation, apparently primarily by uncoupling agonist-activated receptors from their effectors.
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PMID:Sphingosylphosphorylcholine, a naturally occurring lipid mediator, inhibits human platelet function. 1256 68

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-alpha (PKGI-alpha), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-alpha attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-alpha binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of G(q), terminating PAR-1 signaling. Disruption of the RGS-2-PKGI-alpha interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2-/- mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-alpha binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.
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PMID:Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. 1460 79

Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.
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PMID:A platelet secretion pathway mediated by cGMP-dependent protein kinase. 1528 Mar 95

Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP-dependent protein kinase (PKGI) in vascular smooth muscle cells (VSMC), inhibiting agonist-induced intracellular Ca2+ mobilization ([Ca2+]i). The relative roles of the two PKGI isozymes, PKGIalpha and PKGIbeta, in cyclic GMP-mediated inhibition of [Ca2+]i in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+]i in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGIalpha or PKGIbeta were created, and the ability of PKGI isoforms to inhibit [Ca2+]i in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGIalpha or PKGIbeta, 8-Br-cGMP activation suppressed [Ca2+]i by thrombin receptor activation peptide (TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively (p <0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGIalpha- and PKGIbeta-specific antibodies showed PKGIalpha is the predominant VSMC PKGI isoform. [Ca2+]i following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells (Co403). 8-Br-cGMP significantly inhibited TRAP-induced [Ca2+]i in Co403, causing a 4-fold increase in the EC50 for [Ca2+]i. In the absence of 8-Br-cGMP, suppression of PKGIalpha levels by RNA interference (RNAi) led to a significantly greater TRAP-stimulated rise in [Ca2+]i as compared with control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGIalpha expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]i. Adenoviral overexpression of PKGIbeta in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGIalpha expression by RNAi. These results support that PKGIalpha is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.
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PMID:Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells. 1644 62

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myristate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 microM. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 microM, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 microM, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 microM. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 microM NM and was completely inhibited by 50 microM NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.
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PMID:Inhibitory mechanism of human platelet aggregation by nafamostat mesilate. 1680 Oct 94

Proteinase-activated receptor-1 (PAR(1)), a thrombin receptor and the prototype of a newly discovered G-protein-coupled receptor subfamily, plays an important role in tumor development and progression. In this study, we documented the expression of the thrombin receptors PAR(1), PAR(3), and PAR(4) in permanent hepatocellular carcinoma (HCC) cell lines and primary HCC cell cultures. Stimulation of HCC cells with thrombin and the PAR(1)-selective activating peptide, TFLLRN-NH(2), increased transmembrane migration across a collagen barrier. This effect was blocked by the PAR(1) antagonist SCH 79797, confirming that the PAR(1) thrombin receptor subtype is involved in regulating hepatoma cell migration. In addition, the PAR(4)-selective agonist, AYPGKF-NH(2), also stimulated HCC cell migration whilst the PAR(4) antagonist, trans-cinnamoyl-YPGKF-NH(2), attenuated the effect of thrombin on HCC cell migration. PAR(1)- and PAR(4)-triggered HCC cell migration was blocked by inhibiting a number of key mediators of signal transduction, including G proteins of the G(i)/G(o) family, matrix metalloproteinases, ERK/MAPKinase, cyclic AMP-dependent protein kinase, Src tyrosine kinase, and the EGF receptor kinase. Our data point to a cooperative PAR(1)/PAR(4) signaling network that contributes to thrombin-mediated tumor cell migration. We suggest that a combined inhibition of coagulation cascade serine proteinases, the two PARs and their complex signaling pathways may provide a new strategy for treating hepatocellular carcinoma.
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PMID:Thrombin-mediated hepatocellular carcinoma cell migration: cooperative action via proteinase-activated receptors 1 and 4. 1732 77


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