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 a nd extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by protease-activated receptor-1 (PAR-I). a seven-transmembrane G protein-coupled receptor expressed in the nervous s ystem 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 cone s, axons, and dendrites in neurons and processes in astrocytes, The:signal is conveyed by a series of localized ATP-dependent reactions directed to th e actin cytoskeleton. How cells meet the dynamic and localized energy deman ds 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 ATF !:within subcellular compartments. The interaction was confirmed in vitro a nd in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signali ng pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton, Thrombin-stimulated intracellular calcium rele ase 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 cyto skeletal reorganization.