Dynamics and mechanism of p130Cas localization to focal adhesions.
The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.
Donato
Dominique M
DM
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Ryzhova
Larisa M
LM
Meenderink
Leslie M
LM
Kaverina
Irina
I
Hanks
Steven K
SK
D000818:Animals; D000911:Antibodies, Monoclonal; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D017930:Genes, Reporter; D014644:Genetic Variation; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice / Q000196:embryology; D051419:Paxillin / Q000032:analysis / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D014945:Wound Healing / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D050719:Crk-Associated Substrate Protein; D008164:Luminescent Proteins; D051419:Paxillin; D019061:src-Family Kinases