Abstract
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.
Highlights
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K)1 catalyzes the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), a signaling phospholipid, by phosphorylating PI4P at the D-5 position of the inositol ring
KDa splice variant has been shown to contribute to focal adhesion formation in tissue culture cells, the 90-kDa PIP5K␥ might play an important role in platelet actin dynamics
The goal of this study was to investigate the signaling pathway within primary platelets initiated by stimulation of the thrombin receptor and leading to the activation of PIP5K and production of PIP2
Summary
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), a signaling phospholipid, by phosphorylating PI4P at the D-5 position of the inositol ring. Consistent with the role of Rho in PIP5K activation, inactivation of Rho GTPases by Clostridium difficile toxin B reduces cellular PIP2 levels by 50 –90% in several tissue culture cell lines [22, 23]. This results in an inhibition of receptormediated inositol phosphate formation by phospholipase C and PIP2-sensitive phospholipase D and induces profound changes in cell morphology. Rozelle et al [18] have demonstrated that overexpression of PIP5K in tissue culture cells generates actin comets, decreases stress fibers, and suppresses membrane ruffling through pathways that may be mediated by Cdc. There is evidence that PIP5K activity may be regulated by Rho, Rac, or Arf
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