“Cell migration is required and essential for metazoan development, tissue regeneration and repair, as well as in immune surveillance. Many diseases such as cancer, fibrosis, and inherited immunodeficient disorders are associated with altered cell migration,” explained Dr. Saurav Kumar, a postdoctoral fellow in Dr. Jon Cooper’s lab at Fred Hutch.
A critical step in cell migration is the interaction between the cell and the extracellular matrix (ECM). The cell connects to the ECM via the actin cytoskeleton. This connection is mediated by multi-protein complexes called focal adhesions. A critical component of focal adhesions is integrin, a transmembrane-receptor protein. “The mechanism for integrin activation is tightly regulated to ensure proper [cell] migration, but it is frequently disturbed in several diseases including cancer,” said Dr. Kumar.
The activation of integrins exposes the binding sites for ECM outside the cell and integrin-tail-binding sites inside the cell. Inside the cell, integrin-tail-binding sites interact with other proteins to form focal adhesions. Integrin-mediated cell adhesions also activate tyrosine kinase signaling, resulting in tyrosine phosphorylation of integrin-associated proteins. However, the exact role of integrin-activated tyrosine phosphorylation in focal-adhesion formation remained unclear. In a new study published in eLife, Dr. Kumar and colleagues reported that the phosphorylation of a protein called Cas regulates focal adhesion assembly via a positive-feedback loop.
“In our recently published work, we have identified p130Cas/Cas as a regulator of integrin activity,” said Dr. Kumar, lead author of the study. Cas is an adaptor protein previously known to be one of the main substrates of integrin-activated tyrosine phosphorylation. The authors hypothesized that Cas could be a “signaling hub” for focal adhesion assembly. To evaluate the kinetics of Cas recruitment and tyrosine phosphorylation relative to focal adhesion assembly, they endogenously tagged Cas with fluorescence protein in epithelial cell lines (MCF10A and HeLa). This tagged construct allowed the researchers to interrogate the temporal relationship between Cas clusters and other proteins involved in focal adhesion formation, including vinculin and integrin, as well as perform live imaging and cell migration assays.
To interrogate the mechanics of focal adhesion assembly, the researchers utilized state-of-the-art microscopy techniques, including FRET (Fluorescence Resonance Energy Transfer) and TIRF (Total Internal Reflection Fluorescence) imaging. “Previous work done by several groups on fibroblasts plated on fibronectin suggests that the integrin-ECM engagement triggers rapid tyrosine phosphorylation of several proteins,” explained Dr. Kumar. “Surprisingly, using high-speed live-cell TIRF imaging of spreading and migrating epithelial cells, we found that tyrosine phosphorylation of Cas by Src-family kinase (SFK) is pre-requisite for integrin activation and clustering of any tested core focal adhesion proteins.” Many of these microscopy experiments were performed at the Cellular Imaging Shared Resource at Fred Hutch.
The results suggest a two-step model for focal adhesion assembly. “We show that integrin activation comprises two steps: first, phosphorylation of Cas and its recruitment to inactive integrin, which primes the second step of integrin activation and the assembly of other core integrin associated proteins to form mature adhesions,” explained Dr. Kumar. “This first step is amplified by a positive feedback loop involving pCas-Rac1-ROS-SFK, and it is later negatively terminated by the pYCas-specific Cul5-SOCS6 ubiquitin ligase system. The role of Cas in the regulation of integrin activation and clustering is implicated in several cell types, integrin heterodimers and ECM ligands. We show that Cas is involved in regulating integrin activity in bidirectional manners (‘inside out’ as well as ‘outside in’).” This positive feedback loop “drives activation and clustering of integrins and associated proteins during cell spreading and migration,” said Dr. Kumar.
The findings from this study have raised additional questions about Cas and integrin biology. According to Dr. Kumar, these questions include: “What triggers Cas clustering before integrin? Can Cas cluster without integrin? What is the mechanism of pCas-mediated activation of integrin? What is the subcellular localization of initial Cas phosphorylation and clustering? Can lipid nanodomains regulate Cas clustering?” Dr. Kumar and his colleagues in the Cooper Lab are already following up on some of these questions. “We are currently investigating the involvement of lipid domains in the regulation of Cas clustering and vice-versa,” he said. “We are exploring if such interaction between Cas and lipid could induce integrin clustering and activation.”
The spotlighted work was supported by the National Institutes of Health, Fred Hutchinson Cancer Center, and the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Jonathan Cooper contributed to this work.
Kumar S, Stainer A, Dubrulle J, Simpkins C, Cooper JA. 2023. Cas phosphorylation regulates focal adhesion assembly. eLife. 12:e90234.