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

Laboratories of Neurobiology and Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
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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    ABSTRACT: Understanding the mechanisms by which signal transduction pathways mediate changes in RNA abundance requires the examination of the fate of RNA from its transcription to its degradation. Evidence suggests that RNA abundance is partly regulated by post-transcriptional mechanisms affecting RNA decay and this in turn is modulated by some of the same signaling pathways that control transcription. Furthermore, the translation of mRNA is a key regulatory step that is influenced by signal transduction. These processes are regulated, in part, by RNA-binding proteins (RBPs) which bind to sequence-specific RNA elements. The function of RBPs is controlled and co-ordinated by phosphorylation. Based on the current literature we hypothesize that RBPs may be a point of convergence for the activity of different kinases such as phosphoinositide-3-kinase and mitogen-activated protein kinase which regulate RBP localization and function.
    Frontiers in Immunology 12/2012; 3:398. DOI:10.3389/fimmu.2012.00398
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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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    ABSTRACT: Hypoxia-inducible factor-1 (HIF-1) is a well-studied transcription factor mediating cellular adaptation to hypoxia. It also plays a crucial role under normoxic conditions, such as in inflammation, where its regulation is less well understood. The 3'-untranslated region (UTR) of HIF-1α mRNA is among the most conserved UTRs in the genome, hinting toward posttranscriptional regulation. To identify potential trans factors, we analyzed a large compilation of expression data. In contrast to its known function of being a negative regulator, we found that tristetraprolin (TTP) positively correlates with HIF-1 target genes. Mathematical modeling predicts that an additional level of posttranslational regulation of TTP can explain the observed positive correlation between TTP and HIF-1 signaling. Mechanistic studies revealed that TTP indeed changes its mode of regulation from destabilizing to stabilizing HIF-1α mRNA upon phosphorylation by p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2. Using a model of monocyte-to-macrophage differentiation, we show that TTP-driven HIF-1α mRNA stabilization is crucial for cell migration. This demonstrates the physiological importance of a hitherto-unknown mechanism for multilevel regulation of HIF-1α in normoxia.
    Molecular biology of the cell 08/2012; 23(20):4129-41. DOI:10.1091/mbc.E11-11-0949 · 4.47 Impact Factor
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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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    ABSTRACT: The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.
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