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

Abstract

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.

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

“…The phosphorylated hTTP could then be detected by autoradiography and identified by immunoblot with anti-MBP-hTTP antibody. These results show that the purified human TTP is highly phosphorylated in HEK293 cells and that [ 32 P]-labeled TTP is essentially the only labeled phosphoprotein recovered in the purified protein fraction [55].…”

“…Autoradiography and immunoblot results indicate that the truncated hTTP fragments were differentially labeled and expressed at different levels in HEK293 cells [55]. Comparison of the autoradiogram and corresponding immunoblot indicate that multiple phosphorylation sites were present in the N-terminal and the C-terminal regions of hTTP [55]. These analyses provided additional evidence for hyperphosphorylation of human TTP in normal cells [55].…”

“…Comparison of the autoradiogram and corresponding immunoblot indicate that multiple phosphorylation sites were present in the N-terminal and the C-terminal regions of hTTP [55]. These analyses provided additional evidence for hyperphosphorylation of human TTP in normal cells [55]. Using this approach, however, the determination of a specific amino acid residue as being phosphorylated could not be determined.…”

“…HEK293 cells were transfected with the truncated plasmids and labeled with [ 32 P]-orthophosphate. Autoradiography and immunoblot results indicate that the truncated hTTP fragments were differentially labeled and expressed at different levels in HEK293 cells [55]. Comparison of the autoradiogram and corresponding immunoblot indicate that multiple phosphorylation sites were present in the N-terminal and the C-terminal regions of hTTP [55].…”

“…The utility of this approach for the identification of human TTP phosphorylation sites was investigated. The putative phosphorylation sites were selected based on sequence alignment of proteins from mammalian species, including fulllength amino acid sequences of TTP from human, mouse, rat, cow and sheep; and partial amino acid sequences deduced from EST and genomic sequences from chimpanzee, dog, horse and pig [55]. Dozens of serine/threonine to alanine mutant plasmids were constructed to contain a single site mutation or 2-, 3-, 4-, 5-, 6-, 7-or 8-site mutations (Figure 4).…”