Abstract 1640 H2O2-dependent Oxidation of PKA-Ca Differentially Alters its Substrate Selection

Protein kinase A (PKA), a pivotal serine/threonine kinase in cellular signaling, exerts profound influence over various vital processes, including gene regulation, cell cycle progression, metabolism, and apoptosis. Dysregulation of PKA has been linked to a spectrum of diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative disorders. Previous investigations have revealed that oxidation of the alpha isoform of the PKA catalytic subunit (PKA Cα) at C199, located within the P+1 substrate binding region, modulates its reactivity toward the model peptide substrate, Kemptide. Since the P+1 binding site is instrumental in mediating PKA-substrate interactions, we tested the hypothesis that redox modifications on PKA Cα influence its interactions with substrates. Specifically, we used complementary computational and biophysical strategies to assess the impact of oxidation on PKA-ligand interactions. For instance, AutoDock CrankPep (ADCP), a peptide docking program, suggested that sulfenylation of PKA Cα subtly remodels its ligand-binding sites, causing perturbations in interactions with specific ligands while preserving the integrity of others. To evaluate these predictions experimentally, we used surface plasmon resonance (SPR) and fluorescence polarization (FP) spectroscopy to examine the ability of oxidized and reduced forms of PKA to bind different model peptide and protein ligands, including PKI, Kemptide, CREBtide, Crosstide, and GST-CREB. Each interaction was monitored in real-time, yielding apparent rate constants (kon,app, and koff,app) and equilibrium binding constants (KD,app) for every kinase-ligand pairing. Consistent with the ADCP predictions, our findings suggest that oxidation of PKA Cα by H2O2 triggers significant variations in its interactions with select ligands, whereas others remain relatively unaffected. In summation, our comprehensive studies supply valuable insights into the intricate crosstalk between redox and phosphorylation-dependent signaling at the level of kinase-substrate selection. Furthermore, these discoveries significantly impact understanding of kinase regulation in physiological and pathological contexts. This research was funded by National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) Grants #5SC2GM113784 and 1SC1GM130545(to R.H.N.).