Mechanisms of p130Cas-mediated mechano-sensing in cells
Final Report Abstract
The substrate domain of p130Cas, which is located in the central region of p130Cas, is flanked by the amino-terminal SH3 and the carboxy-terminal CCH domains. We and our collaborators in Prague could show that the SH3 and CCH domains are involved in p130Cas localization at focal adhesions, while the substrate domain (SD) itself is not, suggesting that these flanking domains anchor p130Cas molecules to the cytoskeletal complex and that the substrate domain is most likely to be extended upon cytoskeleton stretching. Using an antibody that recognized extended p130Cas SD in vitro, Sawada et al. (2006) observed p130Cas extension in intact cells in the peripheral regions of spreading cells, where higher traction forces are expected and where phosphorylated p130Cas was detected, suggest that the extension and phosphorylation of p130CAS SD is relevant to physiological force transduction. We therefore propose that p130CAS acts as a primary force sensor, i.e. transducing force via mechanical extension and thereby priming phosphorylation and activation of downstream signaling. To verify the proposed model p130CAS-mediated mechano-reception, we determined the contribution of p130CAS in focal adhesion anchoring domains to p130CAS-mediated mechano-reception. For this purpose, we prepared a set of p130CAS constructs including p130CAS mutants which lack the SH3, CCH domain or both of these domains as well as constructs where CCH domain is replaced with either SH3 domain, FAT domain of FAK or LeuZip dimerization motifs. The constructs were expressed in p130CAS-/- cells and localization of all constructs into focal adhesions were verified and quantified. Subsequently, stretch dependent activation of all p130Cas mutants expressed in p130CAS-/- cells were analyzed in the cell stretcher, shear forces were applied to cell populations (1 x 105), and the phosphorylation of p130CAS on tyrosine 410 of p130CAS was determined by Western blot analysis. Taken together, the results from these experiments showed that p130CAS-mediated mechano-transduction is dependent on focal adhesions-anchoring of p130CAS through both SH3 and CCH domains. Our data were complemented with experiments from our collaborators in Prague. Further, p130CAS-mediated mechano-reception will also be analyzed using pERK phosphorylation and/or Rap1-GTPase activation after cell stretching and compared within all cell types. For analysis of Rap1-GTPase activation, Rap1-GTP-specific antibody (New East Biosciences) will be employed.
Publications
- CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics. Cell Mol Life Sci, 71: 727-744, 2014
Janoŝtiak R., Brábek J., Auernheimer V., Tatárová Z., Lautscham L.A., Dey T., Gemperle J., Merkel R., Goldmann W.H., Fabry B., Rösel D
(See online at https://doi.org/10.1007/s00018-013-1450-x)
