Cas phosphorylation regulates focal adhesion assembly

Abstract

Integrins link the cytoskeleton to the extracellular matrix for cell movement. Integrin ligation stimulates tyrosine phosphorylation of integrin-associated proteins but the role of phosphorylation signaling in regulating integrin-cytoskeletal linkages and assembly of focal adhesions is unclear. Using spreading or migrating epithelial cells, we provide evidence that phosphorylated Cas (p130Cas, BCAR1), its binding partner, Crk, and inactive focal adhesion kinase (FAK) cluster together with inactive integrins at the cell periphery at sites that develop into focal adhesions containing F-actin, active integrins, active FAK and mechanosensing proteins such as vinculin and talin. Cas, Crk, Src family kinases (SFK) and Rac1 are required for focal adhesion formation and cell spreading, while vinculin is not needed for Cas clustering. Furthermore, Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest that phosphorylation signaling precedes and regulates mechanosensing during focal adhesion formation.

De Novo Design of Integrin α5β1 Modulating Proteins for Regenerative Medicine

“…We tested if NeoNectin binds cells expressing multiple RGD-interacting integrins in a α5β1-dependent manner using MCF10A Cas mSc cells 21 , a human mammary epithelial cell line expressing endogenously tagged Cas with mScarlet to mark the integrin positive adhesion 22, 23 . Besides α5β1, MCF10A cell line also expresses other RGD binding integrins including αVβ1, αVβ3, αVβ6 24, 25 .…”

“…Besides α5β1, MCF10A cell line also expresses other RGD binding integrins including αVβ1, αVβ3, αVβ6 24, 25 . We first incubated MCF10A Cas mSc cells 21 in presence or absence of 200 nM α5β1-specific antibody mAb16 followed by incubation of 10 nM NeoNectin. The cells were then imaged by Total Internal Reflection Fluorescence (TIRF) microscopy.…”

“…Because of the high affinity and specificity of NeoNectin toward α5β1, we hypothesized that NeoNectin can inhibit α5β1-FN interaction dependent cellular responses as mentioned above. We first tested the effect of soluble NeoNectin on inhibiting spreading of MCF10A epithelial cells, which primarily express α5β1 and α2β1 integrins and can migrate on ECM-containing FN and/or collagen 21 . We first plated MCF10A cells on either collagen- or FN-pre-coated glass surfaces and then treated the cells with NeoNectin at different concentrations for 30 min prior to washes.…”

“…The copyright holder for this preprint this version posted June 25, 2024. ; https://doi.org/10.1101/2024.06. 21.600123 doi: bioRxiv preprint interactions. 5-8 substitutions at designed interacting loop 3 and loop 5 that increased the apparent binding affinity were combined in small libraries which were sorted under stringent conditions (incubation with 0.2 nM biotinylated α5β1 followed by washing and overnight disassociation), yielding 20 optimized designs which were expressed in E. coli and purified (Figures S1G and S1H).…”