Impact of Redox Modification on MAPK Global Substrate Selection

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which "arm" of a branched pathway is activated in response to a given signal. Specifically we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D-recognition site (DRS) and the F-recognition site (FRS). Interestingly, MAPKs contain cysteine residues that are predicted to be oxidized by H2 O2 that lie within or in close proximity to the substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following treatment with various concentrations of H2 O2 . While their activity toward FRS substrates was not changed by H2 O2 pretreatment, both ERK2 and p38α exhibited an increase in the relative activity toward the DRS substrate, Sub-D, at H2 O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2 O2 leads to substantial changes in Km and, to a lesser extent, kcat for both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2 O2 treatment while others exhibited H2 O2 -dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). Likewise, fluorescence polarization assays suggest that oxidation of ERK2 increases its affinity for some ligands while decreasing its affinity for others. Interestingly, the upstream MAPK kinase, MEK1, also recognizes ERK1/2 via interactions with the DRS, suggesting that ERK1/2 activation dynamics may be affected by signal-generated H2 O2 within the cellular environment. Indeed, cell-based assays suggested that pre-treatment of NIH-3T3 cells with the cell permeable H2 O2 scavenger, PEG-catalase, alters ERK1/2 phosphorylation following platelet-derived growth factor (PDGF) treatment. Together, these findings suggest that redox modification of the cysteines located within the MAPK DRS alters the activation dynamics and may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states. © FASEB.