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

Abstract

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.

Figure 1. Tristetraprolin (TTP) is so-named because the deduced amino acid sequences of TTP from mammals contain three repeats with four consecutive proline residues in each repeat

The amino acid sequences used in the alignment include human (NP_003398) [77], mouse (NP_035886) [27], rat (P47973) [78], bovine (P53781) [79], sheep (AY462109) [67], pig (AJ943797, CB288050, CB286240, DY416794 and DY419026) [80], horse (CD536573 and CD536523), chimpanzee (CR555169 andXM_001136016) and dog (AAEX01054372.1 and AAEX01054371.1) [81]. The sequences were aligned with the PILEUP program from GCG. There are 180 amino acid residues in chimpanzee TTP N-terminal region (not shown). The gaps in the horse TTP sequence represent incomplete sequence and it is probably highly similar to the other mammalian TTP sequences. The four praline residues in the three repeats are underlined. The bold residues within the sequence alignment are the phosphorylation sites identified in our study and in other studies and the CCCH residues in the tandem zinc-finger binding motifs. The conserved serine, threonine and tyrosine residues, which correspond to the phosphorylation sites in hTTP identified in our report, are indicated at the top of the sequence alignment. Those corresponding to the previously identified sites in mTTP are indicated at the bottom of the sequence alignment (updated from supplemental table 2 [55]).

Figure 2. Tristetraprolin (TTP) related proteins have been found in many species, ranging from human through yeasts and plants

Sequence identity relative to human proteins: human TTP (100%); mouse TTP (86.2%); rat TTP (86.5%); bovine TTP (87.3%); frog TTP (53.0%); human TIS11B (100%); mouse TIS11B (98.5%); rat TIS11B (98.5%); frog TIS11B (81.8%); human TIS11D (100%); mouse TIS11D (99.6%); frog TIS11D (74.4%). At: Arabidopsis thaliana (weed); Bt: Bovine; Cc: Carp; Ce: Caenorhabditis elegans; Cv: Oyster; Dm: Drosophila; Dr: Zebra fish; Hs: Human; Mm: Mouse; Rn: Norway rat; Rr: Black rat; Sc: Baker’s yeast; Sp: Fission yeast; Xl: Frog.

Figure 3. The tandem CCCH zinc finger sequences are highly conserved among the diverse species

The sequences were aligned with the PILEUP program of GCG software. The vertical shading lines represent the consensus sequence of the tandem CCCH zinc finger sequences as shown at the bottom of the sequence alignment. The bold amino acid residues within the sequence alignment are the CCCH residues in the tandem zinc-finger binding motifs. The amino acid residues in human TTP and homologues are also bolded. The Ce sequences are not used in the alignment because the amino acid residues between the two zinc fingers and within the fingers are relatively divergent from the sequences presented in the figure. A number of CCCH homologues are present in Ce, including PIE-1 (GenBank accession number: AAB17868, germline specification [82]), MEX-5 (GenBank accession number: Q9XUB2, soma/germline asymmetry [83]), MEX-6 (GenBank accession number: Q09436, soma/germline asymmetry [83]), and POS-1 (GenBank accession number: NP_505172): At: Arabidopsis thaliana (weed); Bt: Bovine; Cc: Carp; Ce: Caenorhabditis elegans; Cv: Oyster; Dm: Drosophila; Dr: Zebra fish; Hs: Human; Mm: Mouse; Os: Oryza sativa (rice); Rn: Norway rat; Sc: Baker’s yeast; Sp, Fission yeast; Xl, Frog.

Figure 4. Alignment of amino acid sequences of mammalian TTP (ZFP36), ZFP36L1 (TIS11B), ZFP36L2 (TIS11D), site-directed mutants of human TTP used in our study and the phosphorylation sites identified in our study and in other studies

The amino acid sequences used in the alignment include full-length sequences of TTP from human (NP_003398), mouse (NP_035886), rat (P47973), bovine (P53781) and sheep (AY462109), and from partial amino terminal sequences deduced from the EST sequences of pig (AJ943797, CB288050, CB286240, DY416794 and DY419026), horse (CD536573 and CD536523), chimpanzee (CR555169 and XM_001136016), and dog (BQ172881); full-length sequences of ZFP36L1 (TIS11B) from human (Q07352), mouse (B39590) and rat (NP_058868); and full-length sequences of ZFP36L2 (TIS11D) from human (P47974) and mouse (NP_008818). The sequences were aligned with the PILEUP program of GCG software. The gaps in the horse TTP sequence represent incomplete sequence and it is probably highly similar to the other mammalian TTP sequences. The conserved sequence motifs between the subgroups are boxed and linked with a line between the corresponding boxes. The underlined amino acid residues are the four-proline residues in the three repeats of mammalian TTP. The bold amino acid residues within the sequence alignment are the CCCH residues in the tandem zinc-finger binding motifs; and the conserved serine threonine and tyrosine residues, which correspond to the phosphorylation sites in hTTP identified in our report (indicated at the top of the sequence alignment: S¹², S²¹, S⁴¹, S⁴³, S⁴⁶, S⁴⁸, S⁶⁶, S⁸⁸, S⁹⁰, T⁹², S⁹³, T⁹⁵, T¹⁰⁶, T¹¹¹, Y¹⁵⁸, S¹⁶⁰, S¹⁶⁹, S¹⁸⁴, S¹⁸⁸, T¹⁹⁶, S¹⁹⁷, S²⁰⁷, S²¹⁰, S²¹⁷, S²¹⁸, S²²⁸, S²³⁰, S²³³, T²³⁸, S²⁵², T²⁵⁷, T²⁷¹, S²⁷³, S²⁷⁶, S²⁷⁹, and Y²⁸⁴, S²⁹⁴ and S²⁹⁶); and the three additional phosphorylation sites in mTTP identified previously (also indicated at the top of the sequence alignment: S⁶⁰, S¹¹³ and S³²³ in hTTP, corresponding to S⁵², S¹⁰⁵ and S³¹⁶ in mTTP). The bold phosphorylation sites at the top of the alignment are the conserved phosphorylation sites in TTP, ZFP36L1 and ZFP36L2; which are linked with a line between the two conserved phosphorylation sites. A total of 40 site-directed mutants of His-hTTP were used in our study [55], including 1-site mutation (10 mutants): S88A, S90A, S93A, S186A, S197A, S214A, S218A, S228A, S296A, T271A; 2-site mutation (8 mutants): S(214,218)A, S(214,2280)A, S(214,228)A, S(197, 214)A, S(197, 218)A, S(197,228)A, S(197, 296)A, S(93, 197)A; 3-site mutation (5 mutants): S(88,90,93)A, S(197,214,218)A, S(197,214,228)A, S(197,218,228)A, S(214,218,228)A; 4-site mutation (3 mutants): S(214,218,228,296)A, S(88,90,93,197)A, S(197,214,218,228)A; 5-site mutation (5 mutants): S(88,90,93,214,218)A, S(88,90,93,214,228)A, S(88,90,93,218,228)A, S(197,214,218,228,296)A, S(88,197,214,218,228)A; 6-site mutation (4 mutants): S(88,90,93,214,218,228)A, S(88,197,214,218,228,296)A, S(88,186,197,214,218,228)A, S(88,197,214,218,228)AT271A; 7-site mutation (4 mutants): S(88,186,197,214,218,228,296)A, S(88,197,214,218,228,296)T271A, S(88,90,93,214,218,228,296)A, S(88,90,93,197,214,218,228)A; 8-site mutation (1 mutant): S(88,90,93,197,214,218,228,296)A.