Molecular mechanism of thrombin receptor-specific 33 kDa protein kinase

凝血酶受体特异性33 kDa蛋白激酶的分子机制

• 批准号: 13680711
• 负责人: IDO Masaru
• 金额: $ 0.9万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680711/
• 关键词: Thrombin receptor; Serine; threonine kinase; Platelet; μ-calpain

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.

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。

项目成果

Kume M, Hayashi T, Yuasa H, Tanaka H, Nishioka J, Ido M, Gabazza EC, Kawarada Y and Suzuki K: "Bacterial lipopolysaccharide decreases thrombomodulin expression in the sinusoidal endothelial cells of rats - a possible mechanism of intrasinusoidal microthro

Kume M、Hayashi T、Yuasa H、Tanaka H、Nishioka J、Ido M、Gabazza EC、Kawarada Y 和 Suzuki K：“细菌脂多糖降低大鼠窦内皮细胞中血栓调节蛋白的表达 - 窦内微血栓的可能机制

Shimizu S, Gabazza EC, Taguchi O, Yasui H, Taguchi Y, Hayashi T, Ido M, Shimizu T, Nakagaki T, Kobayashi H, Fukudome K, Tsuneyoshi N, D'Alessandro-Gabazza CN, Izumizaki M, Iwase M, Homma I, Adachi Y and Suzuki K: "Activated Protein C Inhibits the Expressi

Shimizu S、Gabazza EC、田口 O、Yasui H、田口 Y、Hayashi T、Ido M、Shimizu T、Nakagaki T、Kobayashi H、Fukudome K、Tsuneyoshi N、DAlessandro-Gabazza CN、Izumizaki M、Iwase M、Homma

Ido, M.(7人略5人目): "Bacterial lipopolysaccharide decreases thrombomodulin expression on sinusoidal endothelial cells of rats, which may induce liver dysfunction via microthrombus formation in the sinusoids"J. Hepatol.. 38. 9-17 (2003)

Ido，M.（省略 7 人，第 5 人）：“细菌脂多糖降低大鼠肝窦内皮细胞上的血栓调节蛋白表达，这可能通过肝窦中的微血栓形成诱导肝功能障碍”J. Hepatol.. 38. 9-17 (2003) ）

Gabazza EC, Hayashi T, Ido M, Adachi Y and Suzuki K: "Adenosine inhibits thrombin-induced expression of tissue factor on endothelial cells by a nitric oxide-mediated mechanism"Clin Sci. 102. 167-175 (2002)

Gabazza EC、Hayashi T、Ido M、Adachi Y 和 Suzuki K：“腺苷通过一氧化氮介导的机制抑制凝血酶诱导的内皮细胞上组织因子的表达”Clin Sci。

Ido, M.(8人略3人目): "A pivotal role of rho GTPase in the regulation of morphology and function of dendritic cells"J Immunol. 167. 3585-3591 (2001)

Ido, M.（8 篇中的第 3 篇）：“rho GTP 酶在树突状细胞形态和功能调节中的关键作用”J 免疫学杂志 167. 3585-3591 (2001)