Impact of Redox Modification on MAPK Global Substrate Selection

Abstract

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 redox‐sensitive cysteine residues within or in close proximity to their 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 pre‐treatment of the kinases with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pre‐treatment, both ERK2 and p38α exhibited an increase in their relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 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 H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). These findings suggest that redox modification of the cysteines located within the MAPK DRS may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.Support or Funding InformationThis research was supported by the NIH National Institute of General Medical Sciences through grants SC2GM113784 and SC1GM130545 (to RHN) and R01GM119227 (to LBP). Likewise, LM is supported through a Title III HBGI grant from the U.S. Department of Education.