N-WASP-directed actin polymerization activates p130Cas phosphorylation and lamellipodium spreading

Abstract

Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here, we address the mechanism leading to the phosphorylation of CasSD in the context of fibronectin-induced early spreading of fibroblasts. We have previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity but dependent on actin polymerization. Furthermore, we found that CasSD phosphorylation in the early stages of cell spreading required: (1) integrin anchorage and integrin-mediated activation of SFKs, (2) association of Cas with focal adhesion kinase (FAK), and (3) N-WASP-driven actin-assembly activity. These findings, and analyses of the interactions of the Cas domains, indicate that the N-terminus of Cas associates with the FAK-N-WASP complex at the protrusive edge of the cell and that the C-terminus of Cas associates with the immobilized integrin-SFK cluster. Thus, extension of the leading edge mediated by actin polymerization could stretch Cas during early cell spreading, priming it for phosphorylation.

Collective cell migration: guidance principles and hierarchies

“…These include mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), focal adhesion kinase (FAK), phosphoinositide-3-kinase (PI3K)/Akt, Src kinases, Notch, and Rho GTPases. The early activation of these pathways contributes to the intrinsic bipolarity in leader cells [3,[19][20][21][22][23]. As an example, the activation of Rho GTPases (mainly Rac) at the anterior cell part regulates actin polymerization, actomyosin-based contractility, coupling, and force transmission to stabilize integrin-mediated focal adhesions and thus defines leader cell motility [20,24,25].…”

Early Events in Cell Spreading as a Model for Quantitative Analysis of Biomechanical Events

“…Other PTKs cause the stabilization of adhesion components on rigid substrates. SFKs influence actin assembly from adhesions, retrograde flow, adhesion maturation, and adhesion turnover (27,28,(60)(61)(62). Thus, enzymatic pathways are key regulators of the rigidity-sensing mechanisms and could be critical for determining the tissue-specific rigidity response.…”

“…Moreover, several of the adhesion proteins connect transiently (directly or indirectly) to retrograde flowing actin and are repeatedly stretched in this process (53). The resulting exposure of cryptic sites on talin, p130Cas, and other mechanosensitive proteins leads to the activation of signaling pathways to the nucleus or other parts of the cell (53,62,75,76). This signaling presumably involves LIM domain proteins, many of which can shuttle to the nucleus and bind to adhesions in a force-dependent manner (65,77,78).…”

Focal Adhesion Kinase as a Cellular Mechano-Sensor

“…2.5). By moving the virtual spring along the pulling direction with a constant velocity v, the mechanical force F i (t), which is eventually induces protein unfolding, can be detected by using the following equation, 12) where k 0 is the force constant of the spring, z i (t) is the position of force application point, and z spring,i is the position of the spring and given as z spring,i (t) = z i (0) ± ∆z(t), where…”

“…External mechanical force can induce deformation of cell membrane and surface receptors, such as integrin, leading to structural changes in focal adhesions (FAs). FAs are enormous multimolecular complexes located at the periphery of the cell and comprise a number of mechanoresponsive proteins such as integrin [5], talin [6][7][8][9], vinculin [10,11] and p130Cas [12,13]. Through FAs, mechanical signals from the extracellular matrix (ECM) are able to activate the cytoskeleton in the cell [14] and consequently regulate gene expression and cell growth.…”