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

Abstract

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 (GSHbeads or anti-Myc linked to magnetic beads MK21 mediates several p38␣,␤ MAP kinase-dependent processes (for a review, see Ref. 1), demonstrated most clearly by results from targeted disruption of the MK2 gene in mice (2). MK2 (Ϫ,Ϫ) mice have suppressed stress responses. Cellular studies show deficits in motility, chemotaxis, and cytokine production. Macrophages taken from MK2 (Ϫ,Ϫ) mice exhibit normal TNF mRNA induction in response to endotoxin but do not release TNF protein. Cellular TNF protein was markedly decreased in MK2 (Ϫ,Ϫ) macrophages, suggesting a block in production of TNF from TNF mRNA (2). TNF expression is regulated both via mRNA stability and translation (3, 4) but is not completely understood.The p38␣,␤ MAP kinase pathway regulates stability of mRNAs that contain AU-rich elements in their 3Ј-untranslated regions. Examples include TNF, COX-2, interleukin-6, and interleukin-1␤ (4 -6). Evidence chiefly comes from mRNA stabilization caused by transfection of mutationally activated MEK3/MEK6 or by the addition of agents that activate p38 MAPK and conversely from destabilization caused by the addition of a p38␣,␤ MAP kinase inhibitor. Since p38␣,␤ is required to activate MK2, these experiments do not dissect contributions from MK2. Studies in MK2 (Ϫ,Ϫ) cells suggest that MK2 regulates stability of some cytokine mRNAs (2, 4). Lasa et al. (5) first reported that expression of a mutant of MK2 with constitutive activity stabilized COX-2 mRNA in the presence of SB2035780 and that expression of a kinase-defective MK2 blocked the stabilization induced by activated MEK6, arguing that MK2 is necessary and sufficient to induce stabilization of at least the COX-2 mRNA (5). How MK2 regulates cytokine production post-transcriptionally is unknown. Mahtani et al. (7) reported that tristetraprolin (TTP) is an in vitro substrate for MK2, motivating the detailed studies we describe.TTP (for a review, see Ref. 8) destabilizes class II AU-rich elements and is the prototype for a non-zinc finger class of nucleic acid-binding proteins. Destabilization requires integrity of the TTP tandem Cys 3 His RNA binding domains that coordinate zinc in a disklike structure (9 -11). TTP-null mice exhibit many defects including inflammatory arthritis and systemic lupus erythematosis-like symptoms attributed to increased production of TNF (12).TTP is phosphorylated in cells treated with growth factors or cytokines. Phosphorylation occurs at more than one site evident by the appearance of two distinct slower migrating forms of TTP on gels after stimulation that are reversed by phosphatase treatment (7, 13). TTP under...

Anti-inflammatory functions of glucocorticoid-induced genes

“…COX-2, CSF2 and IL-2 genes are dysregulated in the absence of TTP (Carballo et al, 2000;Ogilvie et al, 2005;Phillips et al, 2004), and it seems likely that expression of other inflammatory mediators will also prove to be controlled by TTP. Inflammatory mRNAs can be stabilized via the phosphorylation and inactivation of TTP by MAPK-activated protein kinase 2, a kinase that is activated by p38 MAPK (Carballo et al, 2001;Chrestensen et al, 2004;Stoecklin et al, 2004). At least in cells of the myeloid lineage, this appears to be the principal mechanism for post-transcriptional regulation of inflammatory genes by the p38 MAPK pathway.…”

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

“…Phosphorylation of TTP in the transfected HEK293 cells was further supported by in vivo labeling of proteins with radioactive phosphate, followed by Ni-NTA purification, or by immunoprecipitation with the anti-MBPhTTP serum ( Figure 8A). However, the T271A mutation but not the S186A mutation in hTTP, corresponding to two of the MK2-phosphorylated sites at S264 and S178 of mTTP (23), modestly altered the gel mobility of the mutant protein ( Figure 8A). …”

“…The active form of the MBP-hTTP fusion protein binding to TNF mRNA ARE appears to be in the form of an oligomer rather than a monomer (9). TTP purified from E. coli can be phosphorylated by multiple mitogen-activated protein (MAP) kinases in vitro (9,(20)(21)(22)(23)(24) with kinetics similar to those of other protein substrates (9). Finally, the tandem zinc finger peptide of TTP binds ARE sequences with high affinity (10).…”

“…It is wellknown that TTP is a highly phosphorylated protein in mammalian cells (3,(20)(21)(22)(23)25). Phosphorylation of TTP in mammalian cells partially inhibits its binding to GM-CSF mRNA ARE and enhances its binding to the 14-3-3 protein (20,25).…”

Tristetraprolin Down-Regulates IL-23 Expression in Colon Cancer Cells

“…It has been reported that stimulation with LPS results in a strong and rapid induction of TTP mRNA in mouse macrophages (Mahtani et al, 2001). However, LPS stimulation does not enhance the TTP-mediated degradation of ARE-containing mRNA because it also activates p38 MAPK, which can phosphorylate and inactivate TTP (Chrestensen et al, 2004;Stoecklin et al, 2004). Stimulation with IFN-γ (Sauer et al, 2006), IL-10 (Schaljo et al, 2009) or nicotine (Joe et al, 2011) has been reported to induce TTP expression but not p38 MAPK activity and thus these agents increase the induction of mRNA decay by TTP.…”