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Abstract
Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2–9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.
Highlights
Mechanical stretching of cells causes the substrate domain of p130Cas (CasSD) to be phosphorylated on 15 tyrosine residues embedded along its length
Classical biophysical analyses have determined that CasSD is a typical intrinsically disordered protein, a difficult-to-study group of molecules covering about 30% of human proteins
As it is already known that stretching somehow exposes the tyrosine residues to phosphorylation, a mechanism is proposed where straightening of the p130Cas substrate domain backbone conformation through mechanical stretching can lead to dissociation of p130Cas-binding LIM domain proteins and exposure of CasSD tyrosine residues for phosphorylation
Summary
Total internal reflection fluorescence microscopy and immunofluorescence analysis p130Cas-deficient mouse embryonic fibroblasts expressing p130Cas tagged with GFP (GFP–p130Cas) were grown overnight in DMEM containing 10% FBS on a 50 mg/ml collagen-coated m-Dish (ibidi, Martinsried, Germany) to form a monolayer. The cells were treated with DMSO (0.1%) or 10 mM blebbistatin for 1 hour and scratched by a pipette tip 1.5 hours before fixation. This scratching of the cells simulates wounding of the monolayer. Immunoblotting analysis 1.56106 NIH3T3 cells were allowed to adhere to collagencoated substrates overnight in DMEM containing 10% FBS. The gel was subjected to immunoblotting using anti-pCas-165 and anti-p130Cas (aCas3) antibody to visualize phospho-p130Cas and total p130Cas, respectively
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