MAPKAP Kinase 2 Phosphorylates Tristetraprolin on in Vivo Sites Including Ser super(178), a Site Required for 14-3-3 Binding

• Published in: The Journal of biological chemistry 2004-03, Vol.279 (11), p.10176-10184
• Main Authors: Chrestensen, CA, Schroeder, MJ, Shabanowitz, J, Hunt, D F, Pelo, J W, Worthington, M T, Sturgill, T W
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M310486200

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). MK2[Delta]3B, activated in Escherichia coli by p38 alpha , phosphorylates TTP in vitro at major sites Ser super(52) and Ser super(178) (>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 Ser super(52), Ser super(178), Thr super(250), and Ser super(316) and at SP sites in a cluster (Ser super(80)/Ser super(82)/Ser super(85)). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser super(52) and Ser super(178). Overexpression of MK2 sufficed to increase phosphorylation of Ser super(52) and Ser super(178) but not Ser super(80)/Ser super(82)/Ser super(85) or Thr super(250). Thus, Ser super(52) and Ser super(178) are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.