Mechanical Regulation of PKA Function in Cell Adhesion and Migration

PROJECT SUMMARY Cell adhesion to the extracellular matrix (ECM) is fundamentally important to multicellular life. Focal adhesions (FAs) are the best characterized cellular structures involved in cell-ECM adhesion, and not only serve as physical couplings but are important centers of signal transduction. While FAs are well- known and well-

characterized for being enriched in tyrosine phosphorylated proteins, the phosphoserine and phosphothreonine content of FAs is three times higher than phosphotyrosine. Despite this, the kinases that mediate these modifications and the mechanisms and consequences of their function are poorly understood. We have found

that the cAMP-dependent protein kinase (PKA), a venerable kinase with hundreds of targets throughout the cellular landscape, is present and active within FAs. Moreover, we have found that PKA binds directly to talin, a critically important mechanosensitive FA scaffolding protein, in a manner that requires conformational unfolding

of talin. These observations suggest that talin is a mechanically-gated A-kinase anchoring protein (AKAP) that couples cellular tension to PKA signaling within FAs. Here, we propose to use biophysical, biochemical, and cell biological approaches to characterize the regulation and function of this new axis of localized

mechanotransduction. We will delineate how (1) molecular and cellular mechanics regulate the PKA-talin interaction; (2) PKA-mediated phosphorylation regulates the function and interaction of talin, TAPs, and other FA proteins; (3) disrupting talin-PKA interaction and associated signaling events impact cell adhesion, cell

spreading, and cell migration; and (4) cell adhesion and contractility regulate FA PKA activity. These investigations will not only reveal new facets of adhesion-dependent signal transduction, but will also identify FAs as a new, discrete niche for PKA function and establish a paradigm for mechanical regulation of this

ubiquitous kinase that may play a role in other mechanosensitive cellular structures and events.