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

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). MK2Δ3B, activated in Escherichia coli by p38α, 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, Ser ¹⁷⁸, 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/Ser ⁸²/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.