Impact of Redox Modification on Global Kinase Substrate Selection

Reactive oxygen species (ROS) have recently emerged as critical second messengers in many signaling pathways, including those regulating cell proliferation, migration, differentiation, and gene expression. While much progress has been made in understanding the mechanisms by which ROS levels are modulated 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 oxidation. Indeed, 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. Interestingly, in many cases, the redox-sensitive Cys is located in a region involved in substrate recognition, suggesting that oxidation may alter the substrate preference of a given kinase. To investigate this notion further, we used functional protein microarrays to examine the global substrate selection of several kinase family members under oxidized and reduced states. These studies, together with further biochemical analyses using model peptide substrates, suggest that oxidation leads to differential phosphorylation of many substrates such that distinct sets of substrates are targeted by the oxidized and reduced forms of the kinase. Importantly, in many cases, the redox sensitive Cys in the affected kinases 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.