Pflum, M. K. H. | bioRxiv Science

bioRxiv2025

Chemoproteomic Profiling of PKA Substrates with Kinase-catalyzed Crosslinking and Immunoprecipitation (K-CLIP)

Phosphorylation is a highly regulated protein post-translational modification catalyzed by kinases. Kinases and phosphorylated proteins are key players in a myriad of cellular events, including cell signaling. When cell signaling networks are improperly regulated by kinases, various pathologies can arise, such as cancers and neurodegenerative disease. With critical roles in normal and disease biology, kinase-substrate interactions must be thoroughly characterized. Previously, the chemoproteomic method, kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP), was developed to identify the kinases of a phosphoprotein substrate of interest. Here, K-CLIP was modified to profile the substrates of a kinase of interest. Specifically, the substrate profile of cAMP-dependent protein kinase (PKA) was studied with K-CLIP using a new ATP analog, ATP-alkyne aryl azide. Kinase-focused K-CLIP discovered SMC3 as a PKA substrate. With versatility for any kinase or phosphoprotein substrate of interest, K-CLIP will expand our understanding of kinase-mediated cell biology in healthy and diseased states.

biochemistry↗

bioRxiv2023

Protein Kinase A is Functional Component of Focal Adhesions

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 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.

cell biology↗