Redox regulation of protein kinase function: biochemical mechanisms and cellular consequences

SUMMARY. My research program focuses on understanding the organization and regulation of phosphorylation-dependent signaling networks in health and disease. In particular, we are interested in deciphering the biochemical and cellular mechanisms of crosstalk between phosphorylation-dependent signaling and other signaling pathways at the level of posttranslational modification of protein kinases. One

attractive modification is reversible oxidation, mediated by reactive oxygen species (ROS). ROS are emerging as critical second messengers in signaling pathways related to many pervasive diseases, including diabetes, cancer, and cardiovascular disease. A growing body of evidence suggests that protein kinases are directly

regulated by redox modifications. For instance, the reversible oxidation of Cys residues in redox-sensitive kinases has been shown to influence their activity (both positively and negatively), subcellular localization, and protein-protein interactions. In many cases, the modified Cys is conserved among other members in the same

kinase family. This raises the intriguing possibility that reversible oxidation may be a general means of regulating kinase function inside cells. To explore this possibility further, we are examining the impact of H2O2-depedent oxidation on the global substrate selection of several mitogen-activated protein kinase (MAPK) and AGC family

members using functional protein microarrays and complementary biochemical, biophysical, mutational, computational, and cell-based strategies. Our data suggest that H2O2-dependent oxidation shifts the substrate preference of many kinases, leading to distinct substrate profiles in the oxidized and reduced states. For

instance, with support of an NIGMS SC1 award (5SC1GM130545), we are currently studying the biochemical mechanisms underlying the H2O2-induced shifts in the substrate selection of the canonical AGC and MAPK family members, PKA-Cα and ERK2. These studies suggest that reversible oxidation of PKA-Cα and ERK2

increases their affinity for some substrates while decreasing or having little effect on others. Interestingly, different types of redox modification (e.g., diamide-mediated oxidation, H2O2-dependent oxidation, or glutathionylation) led to different activity profiles toward the same model substrates. With support from the

proposed MIRA, we propose to build on these studies by asking questions about 1) the effects of redox modification of kinase substrate selection in various cellular contexts and their impact on signaling outcomes related to disease; 2) the impact of redox modification on the spatiotemporal regulation of kinase activity profiles

in cells; and 3) how the global substrate profiles of other protein kinases, including understudied kinases in the “dark kinome”, are affected by H2O2-dependent oxidation and/or different types of redox modifications. Together, these studies will offer unique insights into mechanisms of crosstalk between redox- and phosphorylation-

dependent signaling pathways in physiological & pathological processes. Importantly, a MIRA will also facilitate mentoring of trainees from underrepresented minority groups, promoting diversity of the biomedical workforce.