PREFERENTIAL ACTIVATION OF THE P46 ISOFORM OF JNK SAPK IN MOUSE MACROPHAGES BY TNF-ALPHA/

A pleiotropic cytokine, tumor necrosis factor-alpha (TNF alpha), regul ates the expression of multiple macrophage gene products and thus cont ributes a key role in host defense, In this study, we have investigate d the specificity and mechanism of activation of members of the c-Jun- NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfami ly of mitogen-activated protein kinases (MAPKs) in mouse macrophages i n response to stimulation with TNF alpha. Exposure of macrophages to T NF alpha stimulated a preferential increase in catalytic activity of t he p46 JNK/SAPK isoform compared with the p54 JNK/SAPK isoform as dete rmined by: (i) separation of p46 and p54 JNK/SAPKs by anion exchange l iquid 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/JNK K, an upstream regulator of JNK/SAPKs, to phosphorylate recombinant ki nase-inactive p46 and p54 JNK/SAPKs, Endogenous MKK4 was able to trans phosphorylate both isoforms, In addition, both the p46 and p54 JNK/SAP K isoforms were phosphorylated on their TPY motif in response to TNF a lpha stimulation as reflected by immunoblotting with a phospho-specifi c antibody that recognizes both kinases, Collectively, these results s uggest 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 ac tivation within the JNK/SAPK subfamily of MAPKs may be an important me chanism through which TNF alpha regulates macrophage phenotypic hetero geneity and differentiation.