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发挥重要作用。蛋白水解酶激活受体1(PAR1/凝血酶受体)是G蛋白偶联受体(GPCR)的一员，包含7个跨膜区。凝血酶在Arg41/Ser42多肽键上切割受体，并揭开以Ser-Phe-Leu-Leu-Arg-ASN(凝血酶受体激动肽/TRAP)开始的连接的氨基末端序列；随后该配体序列与受体主体的结合与G蛋白介导的跨膜信号耦合。G蛋白偶联受体激酶(GRKs)对GPCRs的磷酸化被认为在受体调节中起着核心作用。最近，酪蛋白激酶I(CKI)被报道可磷酸化GPCR。我们先前在凝血酶刺激的血小板中发现了一种新的33 kDa丝氨酸/苏氨酸蛋白激酶(PK33)，它参与了PAR-1胞浆尾部的磷酸化。在本研究中，我们研究了PK33是否属于GRK或CKI家族。通过HiTrap-DEAE、HiTrap-SP、HiTrap-Blue和HiTrap-Heparin层析，从凝血酶刺激的血小板中部分纯化出PK33。用抗GRK2、抗GRK3、抗GRK5或抗CKI抗体进行Western blotting和凝胶内复性蛋白激酶实验检测肝素-HiTrap-Heparin的活性。GRK2、GRK3和CKI在HiTrap-肝素结合组分中的洗脱方式与PK33不同。GRK5不与HiTrap-Heparin结合。本研究结果表明，PK33是一种不同于GRK2、GRK3、GRK5或CKI的新的蛋白激酶。

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.