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

Abstract

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 proteaseactivated receptor-1 (PAR-1), a seven-transmembrane G proteincoupled 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. P rotease activated receptor-1 (PAR-1) mediates the cellular responses to thrombin during blood coagulation, cell proliferation, vascular permeability changes, tumor metastasis, and nervous system injury (1-3). PAR-1 is a seven-transmembrane G protein-coupled receptor with a novel activation mechanism. Proteolysis at a thrombin cleavage site in the extracellular amino terminus exposes a new amino terminus containing the peptide ligand SFLLRN, which binds intramolecularly to initiate intracellular signals (4). Although originally detected in platelets, endothelial cells, and fibroblasts, PAR-1 is also expressed in the nervous system in a developmentally regulated manner and by specific subpopulations of neurons and astrocytes that are especially vulnerable to neurodegeneration and ischemic injury (1,5,6).In most cells expressing PAR-1, activation of the receptor transmits signals to the actin cytoskeleton that profoundly alter cell shape. Platelets, for example, convert from a spherical to disk shape and extend filopodia (7), endothelial cells contract (8), neurons retract axons, and astrocytes resorb processes and flatten their cell bodies (9-11). These signals also regulate changes in actin-related cell motility observed in neurons (10), fibroblasts (12), and tumor cells (3). The morphological response is mediated by a key signaling pathway that uses serine͞ threonine kinases, G␣12͞13, RhoA, and myosin light chain kinase; actomyosin contraction ...

Thrombin Receptor Signaling to Cytoskeleton Requires Hsp90

“…Cultured astrocytes were incubated overnight in serum-free Dulbecco's modified Eagle's medium and solubilized in immunoprecipitation buffer (50 mM Tris (pH 7.4), 1 mM EGTA, 150 mM NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 g/ml aprotinin, 10 g/ml pepstatin, and 10 g/ml leupeptin) as described earlier (16). The supernatant was immunoprecipitated with either control antibody (rabbit IgG) or anti-PAR-1 antibody generated against the SFLLRN peptide (16) overnight at 4°C in the presence of protein A-Sepharose.…”

“…Morphological Assay-Rat astrocyte cultures were used for morphological studies as described previously (16,26). Briefly, cultures were seeded at a density of 9.6 ϫ 10 5 cells in a 22-mm 12-well plate and grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 10 h. Cultures were rinsed and differentiated in serum-free Dulbecco's modified Eagle's medium containing 1.5 mM dibutyryl cAMP for 2 h; astrocytes developed a stellate morphology.…”

“…Transient transfections were performed for 6 h using 0.5 g of DNA mixed with 4 l of Novafector (Venn Nova, Pompano Beach, FL) according to the manufacturer's protocol. After 18 h, cells were differentiated with 2 mM dibutyryl cAMP in serum-free medium for 6 h. Following addition of thrombin (160 nM) for 2 h, the cells were fixed, and morphology of the fluorescent cells was quantitated (16). Neuronal cells respond to thrombin at low concentrations (10 pM) when grown on plastic dishes, whereas higher amounts (160 nM) of the protease are required when grown on glass coverslips used for the transfection studies.…”

“…Some wells received 1 M U73122 (BIOMOL Research Labs Inc.) for 10 min prior to 10 pM thrombin and 10 mM EDTA injection. The relative intracellular calcium levels were monitored at 488 nm for 210 s in four to eight identically treated wells as described earlier (16).…”

“…On the other hand, PAR-1-mediated activation of RhoA and the subsequent morphological changes involve activation of G␣ 12/13 (3). Recently, we showed that the PAR-1 cytoplasmic tail (C-tail) may be important for the regulation of cytoskeletal changes (16). It is not known how PAR-1 transduces these diverse signals intracellularly.…”