Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP₂ effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase α (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP₂ signaling. PIP5KI overexpression increased PIP₂ and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP₂ and PI4P synthesis in cells. However, Y-27632 had no effect on PIP₂ synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP₂ synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and non-ruffling phenotype. Our results establish the physiological role of PIP₂ in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP₂ in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP₂.