Expression, purification, and biochemical characterization of the antiinflammatory tristetraprolin: a zinc-dependent mRNA binding protein affected by posttranslational modifications

Abstract

Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor alpha mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 microM Zn(2+). The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex.

Figure 1

Purification of MBP–hTTP from E. coli and characterization of anti-MBP–hTTP antibodies. (A) Purification of MBP–hTTP stained with Coomassie blue: lane 1, protein size standards; lane 2, homogenate (50 μg of protein); lane 3, supernatant (50 μg); lane 4, amylose resin fraction (5 μg); lane 5, Superose 12 fraction (2 μg); lane 6, Mono Q fraction (1 μg); and lane 7, MBP eluted from the amylose resin column (5 μg). The positions of MBP–hTTP and MBP are indicated. (B) Detection of MBP–hTTP with anti-MBP serum. The samples were identical to those in panel A except that ∼10% of the amount of protein was used in each lane. (C) Detection limit of anti-MBP–hTTP serum by Western blotting. Nonfusion hTTP purified from E. coli as shown in Figure 7A (lane 2) (1, 5, 10, 15, and 20 ng) as indicated was probed with the anti-MBP–hTTP serum (1:10000) for 1 h and with GAR–HRP (1:10000) for 30 min and exposed to X-ray film for 1 min. (D) Characterization of the anti-MBP–hTTP serum by Western blotting. Protein samples were probed with the anti-MBP–hTTP serum (1:10000) for 1 h and with GAR–HRP (1:10000) for 1 h and exposed to X-ray film for 1 min: lane 1, purified MBP–hTTP (50 ng); lane 2, untransfected HEK293 cell extract (50 μg); lane 3, pBS+ transfection extract (50 μg); lane 4, pHA-hTTP transfection extract (50 μg); lane 5, pHA-mZfp36L1/mTIS11b transfection extract (50 μg); lane 6, pHA-mTTP transfection extract (50 μg); lane 7, transfection extract with full-length hTTP (50 μg); lane 8, transfection extract with amino-terminal residues 1–173 of hTTP (50 μg); and lane 9, transfection extract with carboxyl-terminal residues 97–326 of hTTP (50 μg).

Figure 2

Expression and localization of active TTP in transfected human cells. (A–C) Expression and localization of TTP by immunocytochemistry. HEK293 cells were transfected with pBS+ control plasmid (A) and pHis-hTTP plasmid (B and C). The cells were immunostained with the anti-MBP-hTTP serum (1:5000 dilution) and labeled with goat anti-rabbit Alexa Fluor 488 (1:1000 dilution). Immunofluorescence was recorded with a confocal microscope as either a single image (A and B) or serial images of optical sections of a single cell with a 0.5 μm interval (C). (D) Expression and localization of TTP by immunoblotting. HEK293 cells were transfected with pHis-hTTP. The lysate was centrifuged at 1000g, resulting in S1000g and P1000g. S1000g was further centrifuged at 10000g, resulting in S10000g and P10000g. P1000g was extracted with a buffer containing 0.45 M KCl and centrifuged at 10000g, resulting in the 10000g supernatant (nucleic extract, NE) and the 10000g pellet (NE pellet). Proteins (8 μL) were separated by 10% SDS–PAGE, transferred onto a nitrocellulose membrane, and detected with the anti-MBP–hTTP serum. The position of TTP is indicated. The S10000g lane was loaded with proteins extracted from 10% of the cells used for other samples. (E) ARE binding activity by GMSA. The ARE binding activity was assayed by using proteins prepared as described for panel D. Each assay used 1 μL of each fraction. The positions of TTP–probe complexes and free probes are indicated.

Figure 3

Purification of active TTP from transfected human cells. The 10000g supernatant (lane 2) was mixed with Ni–NTA beads with gentle rotation. The mixtures were then centrifuged at 1000g, resulting in the 1000g supernatant (unbound, lane 3) and the 1000g pellet (beads). The 1000g pellet was washed four times with 20 mM imidazole buffer, resulting in the 1000g supernatants as washes 1–4 (lanes 4–7). Proteins bound to the washed beads were eluted successively with 50, 100, 150, 200, and 250 mM imidazole buffer (lanes 8–12, respectively). (A) Silver staining. Proteins (20 μL) were separated by 10% SDS–PAGE and detected by silver staining. TTP and some of the minor contaminated proteins were also identified by MALDI-TOF MS as indicated. (B) Immunoblotting. Proteins (4 μL/lane) were separated by 10% SDS–PAGE, and TTP was detected with the anti-MBP–hTTP serum. The position of TTP is indicated. (C) Gel mobility shift assay. Each assay used 1 μL of each fraction generated in the Ni–NTA purification process. The positions of TTP–probe complexes and free probes are indicated.

Figure 4

Identification of purified TTP free of endogenous ARE binding activity. HEK293 cells were transiently transfected with pHis-hTTP (0.5 μg) and cotransfected with 4.5 μg of pBS+ (lane 6) or calf thymus DNA (lane 7). As negative controls, cells were also transfected with buffers only (lane 2), pBS+ only (5 μg) (lane 3), or pEF-_XEH encoding a His-tagged Xenopus EH domain of intersectin (0.5 μg) and cotransfected with 4.5 μg of pBS+ (lane 4) or calf thymus DNA (lane 5). Proteins were purified with Ni–NTA beads. (A) Silver staining. Each lane was loaded with 5 μL of the purified proteins eluted with 100 mM imidazole buffer. (B) Immunoblotting. Each lane was loaded with 5 μL of the 10000g supernatant and detected with the anti-MBP–hTTP antibodies. (C) Gel mobility shift assay. Each assay used 4 μL of the 10000g supernatant (lanes 2–5) or 4 μL of the purified proteins in 100 mM imidazole elution buffer (lanes 6–9). The positions of TTP, TTP–probe complexes, and free probes are indicated.

Figure 5

Size of active TTP from transfected human cells. The size of TTP was estimated by size exclusion chromatography using the Superose 6 column and the 10000g supernatant from HEK293 cells transfected with pHis-hTTP (A–C) and using the Superose 12 column and TTP purified by a Ni–NTA affinity column from HEK293T cells transfected with pHis-hTTP (D–F). Fractions were analyzed for the presence of TTP by immunoblotting with anti-MBP–hTTP antibodies (A, D, and E) and analyzed for ARE binging activity by GMSA (B). Lane L in panels A and B represents the initial proteins loaded in the size exclusion columns. The positions of TTP and TTP–probe complexes are indicated. The size of TTP was estimated using a standard curve generated with protein standards run on the same column under identical conditions (C and F), in which K_av = (V_e − V_o)/(V_t − V_o), where V_e, V_o, and V_t are the elution volume of the protein determined by the experiment, the void volume determined with blue dextran (2000 kDa), and the bed volume of the column provided by the manufacturer (Amersham), respectively. The protein standards that were used were provided by the manufacturer (Amersham): bovine pancreas ribonuclease A (13.7 kDa), bovine pancreas chymotrypsinogen (25 kDa), hen egg ovalbumin (43 kDa), bovine serum albumin (67 kDa), rabbit muscle aldolase (158 kDa), bovine liver catalase (232 kDa), horse spleen ferritin (440 kDa), and bovine thyroid thyroglobulin (669 kDa). Similar results were obtained using the Superose 12 column and the same extracts as in panels A–C (data not shown).

Figure 6

Effect of zinc, EDTA, and SDS on the ARE binding activity of TTP. ARE binding activity was assayed by GMSA using TTP (1 μL) purified by Ni–NTA beads and eluted with 100 mM imidazole buffer as described in the legend of Figure 4A (lane 6). The positions of TTP–probe complexes and free probes are indicated. (A) Effect of zinc and EDTA. GMSA buffers contained various concentrations of ZnCl₂, EDTA, or mixtures of ZnCl₂ and EDTA as indicated. (B) Effect of SDS. GMSA buffers contained various concentrations of SDS as indicated.

Figure 7

Binding kinetics of TTP for TNF mRNA ARE. Proteins were diluted with 100 mM imidazole buffer to 8.5 μL before being assayed for ARE binding activity in a 15 μL reaction mixture by GMSA. Gels were exposed to phosphorimager screens, and the signal intensity of the TTP–probe complexes and free probes was analyzed with ImageQuant 5.1. The results represented the means of two to four duplications. The positions of TTP–probe complexes and free probes are indicated. (A) Silver staining of TTP used for the binding kinetic studies. TTP was purified from transfected HEK293 cells and separated by 10% SDS–PAGE (lane 1) [72 ng/μL of a 70% pure sample, 50 ng (1.5 pmoL)⁻¹ (μL of TTP)⁻¹] or purified from overexpressed E. coli cells and separated by 12% SDS–PAGE (lane 2) [600 ng (18 pmoL)⁻¹ μL⁻¹, 100% purity]. (B) Effect of low ARE concentration (10 nM labeled ARE probe) on ARE binding kinetics: (top) TTP from HEK293 cells, (middle) TTP from E. coli cells, and (bottom) plot of the signal intensity of TTP-probe complexes vs the concentration of TTP. (C) Effect of high ARE concentration (10 nM labeled and 300 nM unlabeled ARE probe) on ARE binding kinetics: (top) TTP from HEK293 cells, (middle) TTP from E. coli cells, and (bottom) plot of the signal intensity of TTP–probe complexes vs the concentration of TTP. (D) Effect of ARE concentrations on the binding kinetics of TTP from E. coli cells: (top) 10 nM labeled ARE probe, (middle) 10 nM labeled and 300 nM unlabeled ARE probe, and (bottom) plot of the signal intensity of TTP–probe complexes vs the concentration of TTP.

Figure 8

Phosphorylation of TTP in vivo and in vitro. (A) The 10000g supernatant from HEK293 cells transfected with wild-type pHish-TTP was treated with or without CIAP (lanes 3 and 4) or with or without PNGase F (lanes 5 and 6). Nonfusion hTTP purified from E. coli cells as shown in Figure 7A (lane 2) was used as a size standard for estimating the extent of the dephosphorylation (lane 2). HEK293 cells transfected with the wild-type plasmid were also labeled with [³²P]orthophosphate in vivo. TTP was then purified from the 10000g supernatant of the labeled cells by affinity purification using Ni–NTA beads (lane 9) or by immunoprecipitation using the anti-MBP–hTTP serum (lane 10). The 10000g supernatants from HEK293 cells transfected with two mutant plasmids (S186A and T271A) are also shown (lanes 7 and 8). Proteins were separated by 10% SDS–PAGE, and TTP was detected with anti-MBP–hTTP antibodies (lanes 2–8). The labeled protein was detected by autoradiography (lanes 9 and 10). As a positive control for deglycosylation, fetuin was treated with or without PNGase F under identical conditions and detected with Coomassie blue staining (lanes 11 and 12). (B) Phosphorylation of MBP–mTTP by MAP kinases in vitro. MBP–mTTP (1 μM) purified from E. coli by amylose affinity resin was treated with MAP kinases, including p42/ERK2 (top) and p38 (bottom). Aliquots of the reaction mixtures were taken at various times as indicated and separated by 10% SDS–PAGE. The gels were exposed to X-ray film. A white line on the image highlights the gel mobility shift of the phosphorylated protein. Similar results were obtained with JNK, and with MBP–hTTP as the substrate (data not shown).