Molecular mechanism of thrombin receptor-specific 33 kDa protein kinase

点击翻译按钮获取中文摘要

Thrombin is a multifunctional serine protease that plays an important role in hemostasis and wound healing. Protease-activated receptor 1 (PAR1/ thrombin receptor) is a member of the G protein-coupled receptor (GPCR) containing seven transmembrane domains. Thrombin cleaves the receptor at the Arg41/Ser42 peptide bond and unmasks a tethered amino-terminal sequence beginning with the Ser-Phe-Leu-Leu-Arg-Asn (thrombin receptor agonist peptide/TRAP) ; subsequent binding of this ligand sequence to the body of the receptor is coupled with a transmembrane signaling mediated by G proteins. Phosphorylation of GPCR by G protein-coupled receptor kinases (GRKs) is thought to play a central role in receptor regulation. Recently, casein kinase I (CKI) was reported to phosphorylate GPCR. We previously identified a novel 33-kDa Ser/Thr protein kinase (PK33) in thrombin-stimulated platelets that participates in the phosphorylation of cytoplasmic tail of PAR-1. In the present study, we examined whether PK33 belongs to the same family of GRKs or CKI. PK33 was partially purified from thrombin-stimulated platelets through chromatography using HiTrap-DEAE, HiTrap-SP, HiTrap-Blue, and HiTrap-Heparin. Fractions after HiTrap-Heparin were examined by Western blotting using anti-GRK2, anti-GRK3, anti-GRK5 or anti-CKI antibody and in-gel renaturation protein kinase assay for PK33. GRK2, GRK3 and CKI were eluted in the HiTrap-Heparin-bound fractions differently from PK33. GRK5 did not bind to HiTrap-Heparin. The results of this study suggest that PK33 is a novel protein kinase distinct from GRK2, GRK3, GRK5 or CKI.