Kinase-catalyzed crosslinking: A comparison of ATP-crosslinker analogs.

Protein phosphorylation is catalyzed by kinases to regulate cellular events and disease states. Identifying kinase-substrate relationships represents a powerful strategy to understand cell biology and disease yet remains challenging due to the rapid dynamics of phosphorylation. Over the last decade, several γ-phosphoryl modified ATP analogs containing crosslinkers were developed to covalently conjugate kinases, their substrates, and their associated proteins for subsequent characterization. Here, kinetics and crosslinking experiments demonstrated that the UV-activated analogs, ATP-aryl azide and ATP-benzophenone, offered the most robust crosslinking, whereas electrophilic ATP-aryl fluorosulfate promoted the most effective proximity-enabled crosslinking. The data will guide future applications of kinase-catalyzed crosslinking to study normal and disease biology.

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.

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.

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.

GBT1118, a voxelotor analog, protects red blood cells from damage during severe hypoxia.

A lack of objective metrics in Sickle Cell Disease (SCD) makes it difficult to assess individual patient therapy options or assess the effects of therapy. This is further complicated by mechanisms of action involving multiple interconnected effects, that combine to relieve SCD symptoms. In 2019, based on the increase in hemoglobin concentration observed in the HOPE trial, the Food and Drug Administration approved voxelotor (Oxbryta®, Global Blood Therapeutics) for SCD patients 12 years and older. The main mechanism of action for voxelotor was increased hemoglobin-oxygen affinity, but other mechanisms may apply. In this study, we assessed the effect of GBT1118, an Oxbryta analog, on hypoxia-induced lethal and sub-hemolytic red blood cell (RBC) membrane damage using RBC Mechanical Fragility (MF), a metric of existing membrane damage and prospective hemolysis. RBC MF was measured non-invasively using a proprietary system comprising an electromagnetic bead mill and fiberoptic spectrophotometry detection. Three cycles of severe hypoxia (<5% oxygenated hemoglobin) with follow-up reoxygenation resulted in a significant increase in RBC MF for all SCD (Hb-S >60%) samples. Supplementation with GBT1118 caused no significant changes in pre-hypoxia RBC MF. However, following GBT1118 treatment, cell stability showed significantly less degradation, as evidenced by a significantly smaller RBC MF increase after three cycles of hypoxia-reoxygenation. These findings indicate that GBT1118 prevents hypoxia-induced membrane damage in sickled RBC, in part by alternative mechanisms not associated with induced changes in hemoglobin-oxygen affinity.