Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells.
Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac.
El-Sibai
Mirvat
M
Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Nalbant
Peri
P
Pang
Huan
H
Flinn
Rory J
RJ
Sarmiento
Corina
C
Macaluso
Frank
F
Cammer
Michael
M
Condeelis
John S
JS
Hahn
Klaus M
KM
Backer
Jonathan M
JM
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002277:Carcinoma; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology*; D022081:Cell Surface Extensions / Q000378:metabolism*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014335:Tropomyosin / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C503229:Baiap2 protein, rat; D009419:Nerve Tissue Proteins; D018129:Phosphatidylinositol Phosphates; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; C060974:phosphatidylinositol 3,4,5-triphosphate; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D018631:ras Proteins