Multiple activation mechanisms of p38α mitogen-activated protein kinase

The p38 alpha MAPK participates in a variety of biological processes. Activation of p38 alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38 alpha. In addition to activation by upstream kinases, p38 alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38 alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38 alpha phosphorylation in MEF cells, and we also showed the presence of other p38 alpha activation pathways. We show that TAB1-mediated p38 alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38 alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38 alpha is associated with an similar to 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38 alpha in the similar to 85-kDa complex is independent from MKK3/6 because only phospho-p38 alpha not associated with the disulfide complex was diminished in MKK3/6DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38 alpha phosphorylation in the peroxynitrite-induced similar to 85-kDa complex. Mutagenesis analysis of the cysteines in p38 alpha revealed that no disulfide bond forms between p38 alpha and other proteins in the similar to 85-kDa complex, suggesting it is a p38 alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38 alpha. Therefore, multiple mechanisms of p38 alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.