Molecular mechanism of thrombin receptor-specific 33 kDa protein kinase

凝血酶是一种多功能丝氨酸蛋白酶，在止血和伤口愈合中起重要作用。蛋白酶激活受体1（PAR 1/ thrombin receptor 1，PAR 1/ thrombin receptor 1）是G蛋白偶联受体（G protein coupled receptor，GPCR）的一个成员。凝血酶在Arg 41/Ser 42肽键处切割受体，并揭开以Ser-Phe-Leu-Leu-Arg-Asn（凝血酶受体激动剂肽/TRAP）开始的束缚的氨基末端序列;随后该配体序列与受体体的结合与G蛋白介导的跨膜信号传导偶联。G蛋白偶联受体激酶（GRKs）对GPCR的磷酸化被认为在受体调节中起核心作用。最近，酪蛋白激酶I（CKI）被报道磷酸化GPCR。我们以前确定了一种新的33 kDa丝氨酸/苏氨酸蛋白激酶（PK 33）在凝血酶刺激的血小板，参与磷酸化的PAR-1的胞质尾。在本研究中，我们检查PK 33是否属于同一家族的GRKs或CKI。通过使用HiTrap-DEAE、HiTrap-SP、HiTrap-Blue和HiTrap-Heparin的层析从凝血酶刺激的血小板中部分纯化PK 33。使用抗GRK 2、抗GRK 3、抗GRK 5或抗CKI抗体通过蛋白质印迹和PK 33的凝胶内复性蛋白激酶测定来检查HiTrap-肝素处理后的级分。GRK 2、GRK 3和CKI在HiTrap-肝素结合级分中洗脱，与PK 33不同。GRK 5不与HiTrap-肝素结合。本研究的结果表明，PK 33是一种新的蛋白激酶，不同于GRK 2，GRK 3，GRK 5或CKI。

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.