Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells.
Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) 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 PIP(2) signaling. PIP5KI overexpression increased PIP(2) 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 PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) 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 PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
M
M
Yamamoto
D H
DH
Hilgemann
S
S
Feng
H
H
Bito
H
H
Ishihara
Y
Y
Shibasaki
H L
HL
Yin
D036002:ADP Ribose Transferases / Q000378:metabolism; D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000256:Adenoviridae; D000818:Animals; D001905:Botulinum Toxins; D002308:Cardiolipins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D051345:Destrin; D018260:Gelsolin / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D033862:Iron-Binding Proteins; D008840:Microfilament Proteins / Q000378:metabolism; D008954:Models, Biological; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051304:Profilins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism*; D014161:Transduction, Genetic; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D054460:rho-Associated Kinases; D000096923:Frataxin; D000099149:Ezrin
D036002:ADP Ribose Transferases; D051339:Actin Depolymerizing Factors; D000199:Actins; D001905:Botulinum Toxins; D002308:Cardiolipins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D051345:Destrin; D018260:Gelsolin; D047908:Intracellular Signaling Peptides and Proteins; D033862:Iron-Binding Proteins; D008840:Microfilament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D010982:Platelet-Derived Growth Factor; D051304:Profilins; D017346:Protein Serine-Threonine Kinases; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; D000096923:Frataxin; D000099149:Ezrin; C063007:exoenzyme C3, Clostridium botulinum