Redox-dependent dimerization of p38α mitogen-activated protein kinase with mitogen-activated protein kinase kinase 3

The kinase p38 alpha MAPK (p38 alpha) plays a pivotal role in many biological processes. p38 alpha is activated by canonical upstream kinases that phosphorylate the activation region. The purpose of our study was to determine whether such activation may depend on redox-sensing cysteines within p38 alpha. p38 alpha was activated and formed a disulfide-bound heterodimer with MAP2K3 (MKK3) in rat cardiomyocytes and isolated hearts exposed to H2O2. This disulfide heterodimer was sensitive to reduction by mercaptoethanol and was enhanced by the thioredoxin-reductase inhibitor auranofin. We predicted that Cys-119 or Cys-162 of p38 alpha, close to the known MKK3 docking domain, were relevant for these redox characteristics. The C119S mutation decreased whereas the C162S mutation increased the dimer formation, suggesting that these two Cys residues act as vicinal thiols, consistent with C119S/C162S being incapable of sensing H2O2. Similarly, disulfide heterodimer formation was abolished in H9C2 cells expressing both MKK3 and p38 alpha C119S/C162S and subjected to simulated ischemia and reperfusion. However, the p38 alpha C119S/C162S mutants did not exhibit appreciable alteration in activating dual phosphorylation. In contrast, the anti-inflammatory agent 10-nitro-oleic acid (NO2-OA), a component of the Mediterranean diet, reduced p38 alpha activation and covalently modified Cys-119/Cys-162, probably obstructing MKK3 access. Moreover, NO2-OA reduced the dephosphorylation of p38 alpha by hematopoietic tyrosine phosphatase (HePTP). Furthermore, steric obstruction of Cys-119/Cys-162 by NO2-OA pretreatment in Langendorff-perfused murine hearts prevented the p38-MKK3 disulfide dimer formation and attenuated H2O2-induced contractile dysfunction. Our findings suggest that cysteine residues within p38 alpha act as redox sensors that can dynamically regulate the association between p38 and MKK3.