Regulation of cell adhesion to collagen via beta1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon.
Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by beta1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate beta1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of beta1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase Cvarepsilon bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of beta1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cepsilon is an essential regulatory step for the trafficking and activation of beta1 integrins and cell spreading on collagen.
Kim
Hugh
H
CIHR Group in Matrix Dynamics, University of Toronto, Room 239, 150 College Street, Toronto, Ontario, Canada M5S 3E2. hugh.kim@utoronto.ca <hugh.kim@utoronto.ca>
Nakamura
Fumihiko
F
Lee
Wilson
W
Hong
Claire
C
Pérez-Sala
Dolores
D
McCulloch
Christopher A
CA
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D003285:Contractile Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D064448:Filamins; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D014746:Vimentin / Q000378:metabolism*
D003285:Contractile Proteins; D064448:Filamins; D019012:Integrin beta1; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D014746:Vimentin; D003094:Collagen; D051744:Protein Kinase C-epsilon