Cas phosphorylation regulates focal adhesion assembly
- Fred Hutchinson Cancer Research Center, United States
Abstract
Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated, and associated with its effectors, Crk/CrkL, in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin b1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin b1 is activated and core focal adhesion proteins including vinculin, talin, kindlin and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin b1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.
Data availability
All raw Western blots generated during the study have been included as source files. Matlab code used in the study is uploaded on GitHub (https://github.com/FredHutch/Cas-integrin-paper-2023-Cooper-lab).
Article and author information
Author details
Christopher Simpkins
Funding
Fred Hutchinson Cancer Research Center
- Saurav Kumar
- Amanda Stainer
- Christopher Simpkins
- Jonathan A Cooper
National Institutes of Health
- Saurav Kumar
- Amanda Stainer
- Christopher Simpkins
- Jonathan A Cooper
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Kumar et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Cas phosphorylation regulates focal adhesion assembly
eLife 12:e90234.
https://doi.org/10.7554/eLife.90234
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