Impact of Redox Modification on PKA Substrate Selection

Abstract

Protein kinases regulate multiple signaling pathways involved in health and disease. For instance, cyclic adenosine monophosphate‐dependent protein kinase (PKA) controls a diverse set of cellular functions, including gene expression, cell cycle progression, metabolism, and apoptosis. Dysregulation of PKA has been linked to many diseases, including diabetes, cancer, and neurodegenerative disorders. PKA is a tetramer consisting of two regulatory and two catalytic subunits. Cyclic AMP binding to the regulatory subunits leads to dissociation of the catalytic subunits, unleashing them to phosphorylate serine and threonine residues on their target substrates. Previous studies suggest that the alpha isoform of the PKA catalytic subunit (PKA Cα) is oxidized on C199 within the P+1 substrate binding region. We hypothesized that, by modifying the substrate selectivity of PKA Cα, oxidation may regulate which pathway is activated in response to a given signal. Specifically, we investigated the impact of redox modification on the substrate selection of PKA Cα. To this end, we used fluorescence polarization (FP) spectroscopy to assess the ability of oxidized and reduced forms of PKA Cα to bind a variety of model peptide ligands (e.g., PKI, Kemptide, and CREBtide). The binding of each ligand was monitored under equilibrium binding conditions and the apparent equilibrium binding parameters (i.e., KD,app) were determined for each kinase‐ligand pair. These studies suggest that H2O2‐dependent oxidation of PKA Cα alters its interactions with some ligands while having little effect on others. To gain further insights into the factors driving the differential changes in the interaction between PKA Cα and its ligands, we are currently using surface plasmon resonance (SPR) to measure changes in the apparent rate constants, kon,app and koff,app, as well as KD,app. Together, these studies offer important insights into the mechanisms of crosstalk between redox and phosphorylation‐dependent signaling at the level of kinase substrate selection and have implications for kinase regulation during normal and pathological states. Interestingly, the site of oxidation is highly conserved among AGC kinase family members. Therefore, in the future, we will use a multipronged approach to explore the impact of redox modification on the substrate selection of other AGC family members, such as PKA Cβ and AKT1, in neuronal signaling and the etiology of various diseases.