Abstract
The serine/threonine kinase Rho-kinase was recently identified as a downstream effector of the small GTPase Rho, mediating effects of Rho on the actin cytoskeleton. Also phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) has been implicated in the regulation of actin polymerization. As the synthesis of PI(4,5)P(2) has been suggested to be affected by Rho proteins, we investigated whether Rho-kinase is involved in the control of PI(4,5)P(2) levels. Overexpression of RhoA in HEK-293 cells increased phosphatidylinositol 4-phosphate (PI4P) 5-kinase activity and concomitantly enhanced cellular PI(4,5)P(2) levels, whereas overexpression of the Rho-inactivating C3 transferase decreased both PI4P 5-kinase activity and PI(4,5)P(2) levels. These effects of RhoA could be mimicked by overexpression of wild-type Rho-kinase and of the constitutively active catalytic domain of Rho-kinase, Rho-kinase-CAT. In contrast, a kinase-deficient mutant of Rho-kinase had no effect on PI4P 5-kinase activity. Importantly, the increase in PI4P 5-kinase activity and PI(4,5)P(2) levels by wild-type Rho-kinase, but not by Rho-kinase-CAT, was completely prevented by coexpression of C3 transferase, indicating that the effect of Rho-kinase was under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinase-CAT stimulated PI4P 5-kinase activity. Finally, the increase in PI(4,5)P(2) levels induced by both Rho-kinase-CAT and RhoA was reversed by the Rho-kinase inhibitor HA-1077. Our data suggest that Rho-kinase is involved in the Rho-controlled synthesis of PI(4,5)P(2) by PI4P 5-kinase.
Highlights
Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2)1 is a versatile phospholipid with pleiotropic cellular functions [1]
We examined whether Rho-kinase can modulate the synthesis of PI[4,5]P2
RhoA Increases PI[4,5]P2 Levels in HEK-293 Cells—We previously demonstrated that intoxication of HEK-293 cells with toxin B from C. difficile resulted in a decrease of cellular PI[4,5]P2 levels, indicating that members of the Rho GTPase family are positively involved in the regulation of PI[4,5]P2 synthesis [13, 36]
Summary
Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2)1 is a versatile phospholipid with pleiotropic cellular functions [1]. The increase in PI4P 5-kinase activity and PI[4,5]P2 levels by wild-type Rho-kinase, but not by Rhokinase-CAT, was completely prevented by coexpression of C3 transferase, indicating that the effect of Rho-kinase was under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinaseCAT stimulated PI4P 5-kinase activity. We show that RhoA stimulated PI4P 5-kinase activity, resulting in enhanced cellular PI[4,5]P2 levels in HEK-293 cells.
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