NF-κB1 p105 Negatively Regulates TPL-2 MEK Kinase Activity

Abstract

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-κB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-κB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-κB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells.

ACKNOWLEDGMENTS

S.B. and J.D. contributed equally to this study.

We thank David Baltimore, Anne O'Garra, Alexander Hoffmann, Alain Israel, Mary Holman, Chris Marshall, Antony Symons, and Phillip Tsichlis for reagents used in this study. We are also indebted to Hamish Allen for helpful comments, Matoula Papoutsopoulou for carrying out semiquantitative PCR assay of TPL-2 mRNA, Christopher Atkins for cell sorting, and other members of the Ley laboratory for support during the course of this study.

This work was supported by the UK Medical Research Council, a Boehringer Ingelheim Fonds fellowship to S.B., a European Union Marie Curie fellowship to J.D., and the Arthritis Research Campaign (project grant L0536 to V.L.).