Protein Kinase A is a Functional Component of Focal Adhesions

Abstract

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and, using a targeted biosensor, active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify fifty-three high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3) - a well-established molecular scaffold, regulator of cell migration, and component of focal and fibrillar adhesions - as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein

“…S2 B ). Moreover, V5-RIIα-mTb, like endogenous RIIα subunits ( 24 ), was present in isolated FACS fractions ( Fig. 1 D ) and catalyzed biotinylation of FACS proteins ( Fig.…”

“…6 A ). Recent work has shown that PKA RIIα subunits are retained in FAs in unroofed cells ( 24 ). Moreover, phospho-Ser99 RII, which is part of an epitope that is exposed upon dissociation of the PKA C subunit following enzyme activation and is used as a readout of PKA activity within the anchored holoenzyme ( 64 , 65 ), is enriched in FAs ( 24 ).…”

“…Recent work has shown that PKA RIIα subunits are retained in FAs in unroofed cells ( 24 ). Moreover, phospho-Ser99 RII, which is part of an epitope that is exposed upon dissociation of the PKA C subunit following enzyme activation and is used as a readout of PKA activity within the anchored holoenzyme ( 64 , 65 ), is enriched in FAs ( 24 ). Immunofluorescence analysis showed the localization of both total and active (i.e., phospho-Ser99) RIIα in GFP-positive FAs is significantly reduced in sh-AV/DD-talin HUVECs compared to sh-WT-talin cells ( Fig.…”

“…PKA has also been shown to be both a complex regulator and effector of integrin-mediated adhesion to the extracellular matrix (ECM) ( 11 , 17 – 23 ), although the underlying molecular mechanisms are not fully understood. Recently ( 24 ), we reported the presence of PKA subunits, activity, and novel substrates within focal adhesions (FAs), dynamic, multiprotein junctions formed between the cytoplasmic tails of ECM-bound integrins and actin microfilaments ( 25 ). This positioning means that FAs are subject to actomyosin-generated mechanical forces that alter the conformation of various FA components and thus regulate FA composition and dynamics ( 26 – 28 ).…”

“…The functional connections between PKA and cell adhesion ( 11 , 13 ), the presence of PKA subunits and activity within FAs ( 24 ), and the precept that AKAPs establish and colocalize with PKA activity microdomains ( 35 ) led us to search for potential AKAPs within FAs. In this study, we identify that PKA binds directly to an α-helix in talin that is cryptic and exposed only when the R9 domain unfolds, establishing talin as a mechanically gated AKAP and PKA as a mechanically gated signaling partner for talin.…”