The Impact of Redox Modification on the Global Substrate Selection of PKA C Alpha

Abstract

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), impact many aspects of cellular physiology. Aside from the incidental generation of ROS that occurs during cellular respiration, regulated ROS production by professional ROS‐generating enzymes is now recognized as a key step in redox‐dependent signaling pathways. While our understanding of the mechanisms controlling cellular ROS levels has grown considerably in recent years, less is known about the molecular signaling events that occur downstream of ROS generation.Oxidation of redox‐sensitive Cys residues can directly impact cellular signaling pathways by causing changes in protein‐protein interactions and/or the enzymatic activity of signaling enzymes. For example, recent evidence suggests that protein kinases are directly regulated by oxidation. However, unlike protein tyrosine phosphatases (which are universally inhibited by oxidation), the effect of oxidation on kinase function appears to be more idiosyncratic. Indeed, both oxidation‐induced activation and inactivation have been reported (sometimes for the same kinase).Here, we examined the impact of redox modification of the alpha isoform of the protein kinase A catalytic subunit (PKA Cα). Previous studies demonstrated that diamide‐mediated oxidation of PKA Cα caused a dose‐dependent decrease in kinase activity when the PKA peptide substrate, Kemptide, was used as a substrate. In contrast, we find that little to no inhibition of PKA αC is observed following diamide‐mediated oxidation if Kemptide is replaced by a cocktail of native protein substrates that includes the general kinase substrates histone H3, casein and myelin basic protein. Molecular modeling of the site of oxidation within PKA Cα may offer clues about the mechanism underlying these observations. Indeed, PKA Cα undergoes oxidation on a conserved Cys residue (C199) located within the activation loop. Since oxidation of this residue changes both the size and the charge of the modified Cys residue and the activation loop is important for substrate binding, this modification may disrupt the binding of some substrates while having little effect on others. This raises the intriguing possibility that oxidation‐induced changes may shift the substrate preference of PKA Cα such that distinct subsets of substrates are phosphorylated in the oxidized and reduced states. We are currently investigating this possibility using functional protein microarrays. In addition to diamide, which is a non‐physiological chemical oxidizing agent, we are also investigating the impact of physiologically‐relevant redox modifications, such as H2O2‐mediated oxidation and glutathionylation, on PKA Cα substrate selection. Importantly, the modified Cys residue in PKA Cα is highly conserved among other AGC family members, suggesting that redox modification may be a general means of regulating kinase function within this important kinase family.Together, these studies will offer new insights into the crosstalk between phosphorylation‐ and ROS‐dependent signaling pathways at the level of protein kinase function. They will also lay a foundation for future studies designed to investigate the role of redox‐dependent modulation of kinase activity in physiological and pathological states.Support or Funding InformationThis work was supported by NIH/NIGMS grant 1SC2GM113784‐01 to RHN. LB is supported by the MS‐RISE Program at NC A&T (NIH Grant # 1R25GM076162‐01A1) and JD is supported by the NC A&T LS‐AMP Bridge to the Doctorate Program.