Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis

Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous Studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser(66), Ser(88), Thr(92), Ser(169), Ser(186), Ser(197), Ser(218), Ser(228), Ser(276) and Ser(296) were identified by MS analyses using MALDI (matrix-assisted laser-desorption-ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)-tandem MS and Multidimensional protein identification technology. Mutations of Ser(197), Ser(218) and Ser(228) to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas Mutations at the other sites had little effect oil its gel mobility. Dephosphorylation and in vivo labelling Studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser(197), Ser(218) and Ser(228) are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser(218) and Ser(228)) respectively. The present study has identified Multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further Studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.