Multiple activation mechanisms of p38alpha mitogen-activated protein kinase.
The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha 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 p38alpha. In addition to activation by upstream kinases, p38alpha 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 p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha 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.
Kang
Young Jun
YJ
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
Seit-Nebi
Alim
A
Davis
Roger J
RJ
Han
Jiahuai
J
D000818:Animals; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D007093:Imidazoles / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D048371:MAP Kinase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D048669:MAP Kinase Kinase 6 / Q000235:genetics / Q000378:metabolism; D048688:MAP Kinase Kinase 7 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D011725:Pyridines / Q000378:metabolism; D034622:RNA Interference; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D011725:Pyridines; C099510:TAB1 protein, MAPKKK activator, vertebrate; D030421:Peroxynitrous Acid; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048670:MAP Kinase Kinase 4; D048669:MAP Kinase Kinase 6; D048688:MAP Kinase Kinase 7; C093642:SB 203580