PROJECT SUMMARY Cell interaction with the surrounding extracellular matrix (ECM) controls nearly every major cellular function – including growth, division, survival, shape, and movement. The ECM is connected, vicariously, to the intracellular actin cytoskeleton at focal adhesions (FAs) – multi-protein complexes that assemble and disassemble to dynamically couple actin microfilaments to the cytoplasmic tails of matrix-bound integrins. In addition to their coupling function, FAs also send & receive signals that communicate & control the adhesive state of the cell. Prominent among these signals is reversible protein phosphorylation, with proteins containing and controlling phosphotyrosine being particularly abundant, important, and well-studied in FA biology. This importance notwithstanding, phosphoserine and phosphothreonine modification of FA proteins is far more abundant than phosphotyrosine, but far less studied and understood. Protein Kinase A (PKA) is a ubiquitous and promiscuous Ser/Thr kinase with complex regulatory roles in cell migration. Several observations suggest that PKA may also be important for signaling within FAs. First, inhibition of PKA early during cell attachment alters FA dynamics, increasing FA size and clustering, and decreases cell spreading. Furthermore, PKA subunits as well as a number of putative PKA substrates have been identified in isolated adhesion complexes as well as in published integrin- and/or focal adhesion-associated proteomes. Furthermore, PKA subunits as well as a number of established and putative PKA substrates have been identified in isolated adhesion complexes and in published integrin- and/or focal adhesion-associated proteomes. Using a focal adhesion-targeted PKA biosensor, we have recently shown highly localized and dynamic PKA activity within individual focal adhesions. Finally, using proximity-labelling and complimentary biochemical approaches, we have shown that PKA regulatory (R) subunits closely interact with the archetypal focal adhesion protein talin. We also report that PKA phosphorylates talin as well as the talin-associated proteins (TAPs) RIAM, DLC1, and TES. Based on these observations, we hypothesize that a discrete pool of PKA interacts with talin, modifies talin and TAPs, and enhances FA dynamics. In summary, PKA associates with talin, but the biochemical nature and determinants of this interaction are not known – this is the goal of Specific Aim 1. PKA appears to directly phosphorylate talin, but the sites and effects of this modification are not known - this is goal of Specific Aim 2. PKA appears to directly phosphorylate several talin-associated proteins, but the sites and effects of these modifications are not known – this is the goal of Specific Aim 3. Finally, while talin and TAPs appear to represent an important target cluster for PKA, the consequences of regulating this cluster on cell adhesion, FA dynamics, cell spreading and migration are not known – this is the goal of S...