Preferential activation of the p46 isoform of JNK/SAPK in mouse macrophages by TNF alpha.
A pleiotropic cytokine, tumor necrosis factor-alpha (TNF alpha), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. In this study, we have investigated the specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages in response to stimulation with TNF alpha. Exposure of macrophages to TNF alpha stimulated a preferential increase in catalytic activity of the p46 JNK/SAPK isoform compared with the p54 JNK/SAPK isoform as determined by: (i) separation of p46 and p54 JNK/SAPKs by anion exchange liquid chromatography and (ii) selective immunodepletion of the p46 JNK/SAPK from macrophage lysates. To investigate the level of regulation of p46 JNK/SAPK activation, we determined the ability of MKK4/SEK1/JNKK, an upstream regulator of JNK/SAPKs, to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 was able to transphosphorylate both isoforms. In addition, both the p46 and p54 JNK/SAPK isoforms were phosphorylated on their TPY motif in response to TNF alpha stimulation as reflected by immunoblotting with a phospho-specific antibody that recognizes both kinases. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNF alpha regulates macrophage phenotypic heterogeneity and differentiation.
Chan
E D
ED
Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.
Winston
B W
BW
Jarpe
M B
MB
Wynes
M W
MW
Riches
D W
DW
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D048031:JNK Mitogen-Activated Protein Kinases; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases