Tuesday, May 14, 2024
5/14/2024
PROJECT SUMMARY Reactive oxygen species (ROS) are emerging as critical second messengers in many signaling pathways related to health and disease. While much progress has been made in understanding the mechanisms by which ROS levels are regulated inside cells, less is known about the molecular signaling events that occur downstream of ROS generation. A growing body of evidence suggests that protein kinases are directly regulated by ROS modification. For instance, the reversible oxidation of specific Cys residues in redox- sensitive kinases has been shown to influence their activity (either positively or negatively), subcellular localization, and protein-protein interactions. In many cases, the modified Cys in the affected kinase is conserved among other members in the same kinase family. This raises the possibility that reversible oxidation may be a general means of regulating kinase function inside cells. To explore this possibility further, we recently used functional protein microarrays to examine the impact of oxidation on the global substrate selection of a series of AGC and CMCG kinase family members. These studies suggest that H2O2-dependent oxidation shifts the substrate preference of many kinases, leading to distinct substrate profiles in the oxidized and reduced states. Interestingly, in most cases, both increases and decreases in substrate phosphorylation were observed. As a consequence, reversible oxidation may play an important role in controlling the signaling specificity of redox-sensitive kinases in cells. To investigate these questions further, we propose 1) to examine the molecular mechanisms underlying the H2O2-induced shifts in kinase substrate selection (Aims 1 & 2) and 2) to begin to explore the functional consequences of redox modification on kinase-dependent signaling processes inside the cell (Aim 3). During these studies, we will focus on two representative AGC and CMGC family members, namely PKA and ERK2. Not only will this provide new insights into the redox regulation of these important kinases, but it will also lay a foundation for the analysis of other redox sensitive kinases identified in the microarray experiments. Together, these studies will offer unique insights into ROS-mediated regulation of kinase function and provide the foundation for future studies into crosstalk between ROS- and phosphorylation-dependent signaling pathways in physiological and pathological states.
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