Creatine kinase, an ATP-generating enzyme, is required for thrombin receptor signaling to the cytoskeleton

Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by protease-activated receptor-1 (PAR-1), a seven-transmembrane G protein-coupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells. Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes. The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton. How cells meet the dynamic and localized energy demands during signal transmission is unknown. Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments. The interaction was confirmed in vitro and in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton. Thrombin-stimulated intracellular calcium release was not affected. Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization.

凝血酶（Thrombin）可调控血管系统损伤后以及血液外渗至周围组织时的细胞事件。凝血酶的病理生理应答由蛋白酶激活受体1（protease-activated receptor-1, PAR-1）介导，该受体是一种在神经系统中表达的七次跨膜G蛋白偶联受体，与血小板、成纤维细胞及内皮细胞中的凝血酶受体完全一致。一旦被凝血酶激活，PAR-1可诱导细胞形态发生快速且显著的变化，尤其表现为神经元内生长锥（growth cones）、轴突（axons）与树突（dendrites）的回缩，以及星形胶质细胞（astrocytes）突起的收缩。该信号通过一系列靶向肌动蛋白细胞骨架（actin cytoskeleton）的局域性ATP依赖性反应进行传递。目前尚不清楚细胞如何在信号传递过程中满足动态且局域化的能量需求。本研究借助酵母双杂交系统（yeast two-hybrid system），发现PAR-1的胞质尾区与肌酸激酶（creatine kinase）的脑型同工酶存在相互作用；肌酸激酶是一种关键的ATP生成酶，可在亚细胞区室（subcellular compartments）内调控ATP水平。该相互作用已在体外（in vitro）与体内（in vivo）得到验证。降低肌酸激酶的表达水平或其ATP生成潜能，可抑制PAR-1介导的细胞形态变化，以及一条以RhoA激活为核心的PAR-1信号通路，RhoA是一种可向细胞骨架传递信号的小G蛋白（small G protein）。凝血酶刺激引发的细胞内钙释放并未受到影响。本研究结果表明，肌酸激酶可结合于PAR-1，从而在细胞骨架重排过程中，按需为受体高效的信号转导（signal transduction）提供位点特异性的高能磷酸基团。