Focal Adhesion Kinase Signaling Spatially Regulates Adhesion Dynamics in Fibroblasts

A key step in cell migration is the dynamic formation and disassembly of adhesions at the front and the concomitant movement and release of adhesions in the rear of the cell. In this thesis we present the evidence that the signals regulating adhesion dynamics in the rear of the cells differ from the signals that regulate adhesion dynamics in the front. The first part of the work presented in this dissertation demonstrates that focal adhesion kinase (FAK) signaling to specific regulators of Rho GTPase controls adhesion movement and trailing edge retraction in fibroblasts. We present a model in which addition of Lysophosphatidic acid (LPA) induced the movement of adhesions and retraction of the trailing edge, thus mimicking tail retraction in migrating cells. FAK, guanine nucleotide exchange factors (GEFs) for Rho and the Rho effector, Rho Kinase II (ROCKII) are critical for the regulation of adhesion movement and trailing edge retraction. Down-regulation of FAK by small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs) blocked LPA induced adhesion movement and trailing edge retraction. This phenotype was rescued by ectopic expression of PDZ-RhoGEF or a Rho effector domain mutant that activates ROCK. The knockdown of PDZ-RhoGEF or ROCK II inhibited LPA-induced trailing edge retraction and adhesion movement. Moreover, over expressed PDZ-RhoGEF co-immunoprecipitated with FAK and localized to FAK containing adhesions. These studies support a model in which FAK and PDZ- RhoGEF co-operate to induce Rho/ROCKII-dependent adhesion movement and trailing edge retraction in response to LPA. In the second part of this dissertation, we demonstrate the importance of a specific scaffold in targeting the MAP kinase pathway to adhesion structures found in the 2 areas of active protrusions (front). We show that the RACK1 (Receptor Activated Kinase C) scaffold protein specifically functions in integrin-mediated activation of the MAPK/ERK cascade and targets active ERK to adhesions present in the lamellipodia of protruding cells. We found that RACK1 associates with the core kinases of the ERK pathway, Raf, MEK and ERK, and that attenuation of RACK1 expression resulted in a decreased ERK activity in response to adhesion. RACK1 silencing also caused a reduction of active ERK in adhesions and decreased the rate of adhesion disassembly in the areas of active protrusions. Our data further indicate that FAK is an upstream activator of the RACK1/ERK pathway in the regulation of adhesion disassembly in the lamellipodia. We suggest that RACK1 tethers the ERK pathway to adhesions found in the lamellipodia and channels signals from upstream activation by integrins to downstream targets at these sites. In summary, the data presented in Chapters 2 and 3 argue that signaling pathways regulating adhesion dynamics within the leading edge of migrating cell differ from the signals that regulate adhesion dynamics at the trailing edge of the cell. We propose that FAK is a major component in coupling the MAP kinase pathway adhesion disassembly in the leading edge and Rho pathway to adhesion movement at the trailing edge.

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