Preferential Activation of the p46 Isoform of JNK/SAPK in Mouse Macrophages by TNFα

• Published in: Proceedings of the National Academy of Sciences - PNAS 1997-11, Vol.94 (24), p.13169-13174
• Main Authors: Chan, Edward D., Winston, Brent W., Jarpe, Matthew B., Wynes, Murry W., David W. H. Riches
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Bacteriophages; Biological Sciences; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Catalysis; Catalytic activity; Cells, Cultured; Cellulose nitrate; Chromatography, Ion Exchange; Enzyme Activation; Gene expression regulation; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Liquid chromatography; Macrophages; Macrophages - drug effects; Macrophages - enzymology; Mice; Mitogen-Activated Protein Kinases; Phosphorylation; Protein isoforms; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.24.13169

A pleiotropic cytokine, tumor necrosis factor-α (TNFα ), 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 TNFα . Exposure of macrophages to TNFα 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 TNFα 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 TNFα regulates macrophage phenotypic heterogeneity and differentiation.