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Published online 5 March 2001. doi:10.1083/jcb.152.5.867
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© The Rockefeller University Press, 0021-9525/2001//867 $5.00
The Journal of Cell Biology, Volume 152, Number 5, , 2001 867-876

Original Article

Phosphatidylinositol 4,5-Bisphosphate Induces Actin Stress-Fiber Formation and Inhibits Membrane Ruffling in Cv1 Cells



Masaya Yamamotoa, Donald H. Hilgemanna, Siyi Fenga, Haruhiko Bitob, Hisamitsu Ishiharac, Yoshikazu Shibasakic, and Helen L. Yina

a Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
b Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, TOREST-JST, Japan
c Department of Metabolic Diseases, University of Tokyo, Tokyo, Japan
Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390.214-6487891214-6487967

helen.yin{at}utsouthwestern.edu

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 {alpha} (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 nonruffling 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.

Key Words: phosphatidylinositol 4,5 bisphosphate • Rho • Rho-dependent serine/threonine kinase • gelsolin • phosphatidylinositol phosphate 5-kinase

© 2001 The Rockefeller University Press

Abbreviations used in this paper: ADF, actin depolymerizing factor; BDM, 2,3-butanedione 2 monoxide; ERM, ezrin/radixin/moesin; HA, hemagglutinin; HSP, high-speed pellet; HSS, high-speed supernatant; LSP, low-speed pellet; PI4P, phosphatidylinositol 4 phosphate; PIP2, phosphatidylinositol 4,5 bisphosphate; PIP5KI, type I phosphatidylinositol phosphate 5-kinase {alpha}; PS, phosphatidylserine; ROCK, Rho-dependent serine/threonine kinase; TLC, thin layer chromatography.


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