Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets.
Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCdelta did not occur in Galpha(q)-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5'-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ', N '-tetraacetic acid), suggesting a role for Galpha(q) pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCdelta. Inhibition of tyrosine phosphorylation or the kinase activity of PKCdelta dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCdelta in a calcium- and Src-family kinase-dependent manner in platelets, with functional implications in thromboxane A2 generation.
Murugappan
Swaminathan
S
Department of Physiology, Temple University School of Medicine, Rm 224, OMS, 3420 N Broad St, Philadelphia, PA 19140, USA.
Shankar
Haripriya
H
Bhamidipati
Surya
S
Dorsam
Robert T
RT
Jin
Jianguo
J
Kunapuli
Satya P
SP
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood* / Q000737:chemistry; D051745:Protein Kinase C-delta; D011760:Pyrrolidinones / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000097:blood; D018179:Receptors, Thrombin / Q000097:blood; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D004963:Estrenes; D011760:Pyrrolidinones; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D013912:Threonine; D014443:Tyrosine; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; C497259:Prkcd protein, mouse; D019061:src-Family Kinases; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D010738:Type C Phospholipases; D013917:Thrombin; C112908:protease-activated receptor 4; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid