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

Duke University, Durham, North Carolina, United States
Biochemical Journal (Impact Factor: 4.4). 03/2006; 394(Pt 1):285-97. DOI: 10.1042/BJ20051316
Source: PubMed


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 Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 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 Ser197, Ser218 and Ser228 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 on 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. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) 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.

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    • "Furthermore, another study reported that in U87 glioblastoma cells, the regulation of cyclin D1 and c-Myc mRNA stability by TTP is controlled by p38 in a PKB-dependent manner (Marderosian et al., 2006), implying interdependent roles for p38 and PKB. Data from phospho-proteomic (Cao et al., 2006, 2007; Navarro et al., 2011) and in vitro (Chrestensen et al., 2004) studies suggest that MK2 and PKB phosphorylate TTP at Ser52/178 and Ser248 respectively (Table 2). "
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    • "These results suggest that PMA-induced differentiation of THP-1 cells activates TTP at multiple levels. Moreover, among p38 MAPK, MK2, and PKC, it has been predicted that TTP bears at least 10 high-probability in vivo sites for phosphorylation, which are potential sites for further kinases such as protein kinase A and B, glycogen synthase kinase-3, c-Jun N-terminal kinase, and extracellular signalregulated kinases 1 and 2 (Cao et al., 2006). How these kinases (alone or in combination) influence TTP function regarding mRNA binding and modulation of mRNA fate remains to be elucidated. "
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    • "TTP family proteins contain a high percentage of serines and threonines, which can undergo phosphorylation 9,10. Phosphorylation of TTP and ZFP36L1 can regulate their subcellular localization, protein stability, and mRNA destabilization function 11-13. "
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