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J. Biol. Chem., Vol. 279, Issue 11, 10176-10184, March 12, 2004

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MAPKAP Kinase 2 Phosphorylates Tristetraprolin on in Vivo Sites Including Ser¹⁷⁸, a Site Required for 14-3-3 Binding^*

Carol A. Chrestensen, Melanie J. Schroeder¶||, Jeffrey Shabanowitz¶, Donald F. Hunt¶, Jared W. Pelo**, Mark T. Worthington**, and Thomas W. Sturgill¶¶

From the Departments of Pharmacology, ^¶Chemistry, Pathology and the ^**Digestive Health Center of Excellence, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3'-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads). MK23B, activated in Escherichia coli by p38, phosphorylates TTP in vitro at major sites Ser⁵² and Ser¹⁷⁸ (>10-fold in abundance) as well as at several minor sites that were detected after enriching for phosphopeptides with immobilized metal affinity chromatography. MK2 phosphorylation of TTP creates a functional 14-3-3 binding site. In cells, TTP was phosphorylated at Ser⁵², Ser¹⁷⁸, Thr²⁵⁰, and Ser³¹⁶ and at SP sites in a cluster (Ser⁸⁰/Ser⁸²/Ser⁸⁵). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser⁵² and Ser¹⁷⁸. Overexpression of MK2 sufficed to increase phosphorylation of Ser⁵² and Ser¹⁷⁸ but not Ser⁸⁰/Ser⁸²/Ser⁸⁵ or Thr²⁵⁰. Thus, Ser⁵² and Ser¹⁷⁸ are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.

Received for publication, September 22, 2003 , and in revised form, December 4, 2003.

^* This work was supported in part by the United States Public Health Service, National Institutes of Health Grants GM62890 (to T. W. S.), GM37537 (to D. F. H.), and DK60720 (to M. T. W.). 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.

Supported by National Institutes of Health Training Grant T32-DK07320.

Both authors contributed equally to this work.

^|| This work was completed in partial fulfillment of the requirements for the Ph.D. degree for this author (University of Virginia, Charlottesville, VA).

^¶¶ To whom correspondence should be addressed: Box 800735, Dept. of Pharmacology, University of Virginia, Charlottesville, VA 22908. Tel.: 434-924-8659; Fax: 434-924-5207; E-mail: tws7w{at}virginia.edu.

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