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Activation of MTK1/MEKK4 by GADD45 through Induced N-C Dissociation and Dimerization-Mediated trans Autophosphorylation of the MTK1 Kinase Domain ^,

Zenshi Miyake,^1,2, Mutsuhiro Takekawa,^1,2,* Qingyuan Ge,³ and Haruo Saito^1,2*

Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,¹ Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,² Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, Massachusetts 01923³

Received 4 August 2006/ Returned for modification 3 October 2006/ Accepted 9 January 2007

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45/ß/). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.

Published ahead of print on 22 January 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

These authors contributed equally to this work.

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