RACK1 regulates integrin-mediated adhesion, protrusion, and chemotactic cell migration via its Src-binding site.
Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the beta integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.
Cox
Elisabeth A
EA
Departments of Pediatrics and Pharmacology, University of Wisconsin, Madison 53706, USA.
Bennin
David
D
Doan
Ashley T
AT
O'Toole
Timothy
T
Huttenlocher
Anna
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002448:Cell Adhesion; D002465:Cell Movement; D002633:Chemotaxis; D003001:Cloning, Molecular; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D019204:GTP-Binding Proteins; D005822:Genetic Vectors; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism / Q000502:physiology*; D051419:Paxillin; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D013997:Time Factors; D014162:Transfection; D016596:Vinculin / Q000737:chemistry
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D005353:Fibronectins; D016023:Integrins; D009363:Neoplasm Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D016596:Vinculin; D019204:GTP-Binding Proteins