Filamin A is required for vimentin-mediated cell adhesion and spreading.
Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface beta1 integrin expression, and beta1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and beta1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-epsilon. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-epsilon, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-epsilon, thereby enabling vimentin phosphorylation, which is important for beta1 integrin activation and cell spreading on collagen.
Kim
Hugh
H
Canadian Institutes of Health Group in Matrix Dynamics, University of Toronto, Toronto, Ontario, M5S 3E2, Canada. hugh.kim@utoronto.ca
Nakamura
Fumihiko
F
Lee
Wilson
W
Shifrin
Yulia
Y
Arora
Pamela
P
McCulloch
Christopher A
CA
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D064448:Filamins; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D019012:Integrin beta1 / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D003285:Contractile Proteins; D064448:Filamins; D007211:Indoles; D019012:Integrin beta1; D008301:Maleimides; D008840:Microfilament Proteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D014746:Vimentin; D003094:Collagen; C497255:PRKCE protein, human; D051744:Protein Kinase C-epsilon; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; C088060:bisindolylmaleimide