Protein kinase A is a 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 53 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 a 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.

Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase-Substrate Pairs.

Phosphorylation of proteins by kinase enzymes is a post-translational modification involved in a myriad of biological events, including cell signaling and disease development. Identifying the interactions between a kinase and its phosphorylated substrate(s) is necessary to characterize phosphorylation-mediated cellular events and encourage development of kinase-targeting drugs. One method for substrate-kinase identification utilizes photocrosslinking γ-phosphate-modified ATP analogues to covalently link kinases to their substrates for subsequent monitoring. Because photocrosslinking ATP analogues require UV light, which could influence cell biology, we report here two ATP analogues, ATP-aryl fluorosulfate (ATP-AFS) and ATP-hexanoyl bromide (ATP-HexBr), that crosslink kinase-substrate pairs via proximity-mediated reactions without the need for UV irradiation. Both ATP-AFS and ATP-HexBr acted as cosubstrates with a variety of kinases for affinity-based crosslinking, with ATP-AFS showing more robust complexes. Importantly, ATP-AFS promoted crosslinking in lysates, which demonstrates compatibility with complex cellular mixtures for future application to kinase-substrate identification.

Protein Kinase A is a 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.

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase.

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

Kinase-Catalyzed Crosslinking and Immunoprecipitation (K-CLIP) to Explore Kinase-Substrate Pairs.

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).