Identification of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of protease-activated receptor 1 (thrombin receptor) in human platelets

Stimulation of human platelets with thrombin or thrombin receptor agonist p eptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the p rotease-activated receptor 1 (PARI). 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 proce dure based on the ability of protein kinase to undergo renaturation and pho sphorylate a protein substrate fixed in a gel, We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplas mic tail of PARI (Pro368-Thr425)(GST-PAR 1) 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 hirudi n-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, sugg esting that it is activated through PAR1. Furthermore, treatment of platele ts with thromboxane A(2) analog, STA(2), led to an activation of this prote in kinase and phosphorylation of PAR1. In conclusion, the present study pro vides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.