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J. Biol. Chem., Vol. 279, Issue 50, 52117-52123, December 10, 2004

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Phosphorylation of Threonine 290 in the Activation Loop of Tpl2/Cot Is Necessary but Not Sufficient for Kinase Activity^*

Brenda S. Luciano, Sang Hsu, Padma L. Channavajhala, Lih-Ling Lin, and John W. Cuozzo

From the Department of Molecular Inflammation, Inflammation Signaling, Wyeth Research, Cambridge, Massachusetts 02140

Cot/Tpl2/MAP3K8 is a serine/threonine kinase known to activate the ERK, p38, and JNK kinase pathways. Studies of Tpl2 knock-out mice reveal a clear defect in tumor necrosis factor- production, although very little detail is known about its regulation and the signaling events involved. In the present study we demonstrated that phosphorylation of Cot was required for its maximal activity as phosphatase treatment of Cot decreased its kinase activity. The Cot sequence contains a conserved threonine at position 290 in the activation loop of the kinase domain. We found that mutation of this residue to alanine eliminated its ability to activate MEK/ERK and NF-B pathways, whereas a phosphomimetic mutation to aspartic acid could rescue the ability to activate MEK. Thr-290 was also required for robust autophosphorylation of Cot. Antibody generated to phospho-Thr-290-Cot recognized both wild-type and kinase-dead Cot, suggesting that phosphorylation of Thr-290 did not occur through autophosphorylation but via another kinase. We showed that Cot was constitutively phosphorylated at Thr-290 in transfected human embryonic kidney 293T cells as well as human monocytes as this residue was phosphorylated in unstimulated and lipopolysaccharide-stimulated cells to the same degree. Treatment with herbimycin A inhibited Cot activity in the MEK/ERK pathway but did not inhibit phosphorylation at Thr-290. Together these results showed that phosphorylation of Cot at Thr-290 is necessary but not sufficient for full kinase activity in the MEK/ERK pathway.

Received for publication, April 2, 2004 , and in revised form, September 16, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Wyeth Research, 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-665-5477; Fax: 617-665-5499; E-mail: jcuozzo{at}wyeth.com.

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