Phosphorylation of TPL-2 on Serine 400 Is Essential for Lipopolysaccharide Activation of Extracellular Signal-Regulated Kinase in Macrophages

Abstract

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-κB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1^−/− macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1^−/− macrophages. TPL-2^S400A expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8^−/− macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-κB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.

We thank Alain Israel for reagents used in this study. We are also grateful to Ben Seddon and Antony Symons (Division of Immune Cell Biology, NIMR) for critical readings of the manuscript and to NIMR Biological Services, NIMR Photographics, and other members of the Ley laboratory for their support during the course of this work.

Work in the laboratory of S.C.L. was supported by the UK Medical Research Council and the Association for International Cancer Research (project grant 03-297 to A.K.). Work on the regulation of TPL-2 kinase in the P.N.T. laboratory was supported by NIH grant RO1CA095431.