Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets.
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the rate-limiting step in the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a signaling phospholipid that contributes to actin dynamics. We have shown in transfected tissue culture cells that PIP5K translocates from the cytosol to the plasma membrane following agonist-induced stimulation of Rho family GTPases. Nonetheless, it is unclear whether Rho GTPases induce PIP5K relocalization in platelets. We used PIP5K isoform-specific immunoblotting and lipid kinase assays to examine the intracellular localization of PIP5K in resting and activated platelets. Using differential centrifugation to separate the membrane skeleton, actin filaments and associated proteins, and cytoplasmic fractions, we found that PIP5K isoforms were translocated from cytosol to actin-rich fractions following stimulation of the thrombin receptor. PIP5K translocation was detectable within 30 s of stimulation and was complete by 2-5 min. This agonist-induced relocalization and activation of PIP5K was inhibited by 8-(4-parachlorophenylthio)-cAMP, a cAMP analogue that inhibits Rho and Rac. In contrast, 8-(4-parachlorophenylthio)-cGMP, a cGMP analogue that inhibits Rac but not Rho, did not affect PIP5K translocation and activation. This suggests that Rho GTPase may be an essential regulator of PIP5K in platelets. Consistent with this hypothesis, we found that C3 exotoxin (a Rho-specific inhibitor) and HA1077 (an inhibitor of the Rho effector, Rho-kinase) also eliminated PIP5K activation and trafficking into the membrane cytoskeleton. Thus, these data indicate that Rho GTPase and its effector Rho-kinase have an intimate relationship with the trafficking and activation of platelet PIP5K. Moreover, these data suggest that relocalization of platelet PIP5K following agonist stimulation may play an important role in regulating the assembly of the platelet cytoskeleton.
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Seun-Ah
SA
Yang
Christopher L
CL
Carpenter
Charles S
CS
Abrams
D000199:Actins / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology* / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D021381:Protein Transport; D013917:Thrombin / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000502:physiology*; D054460:rho-Associated Kinases
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D020741:rho GTP-Binding Proteins