©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Tristetraprolin, a Potential Zinc Finger Transcription Factor, by Mitogen Stimulation in Intact Cells and by Mitogen-activated Protein Kinase in Vitro(*)

Gregory A. Taylor (§) , Michael J. Thompson (¶) , Wi S. Lai (**) , Perry J. Blackshear (§§)

From the (1) Howard Hughes Medical Institute Laboratories, and Section of Diabetes and Metabolism, Division of Endocrinology, Metabolism and Nutrition, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tristetraprolin (TTP) is a potential transcription factor that contains three PPPPG repeats and two putative CCCH zinc fingers. TTP is encoded by the early response gene Zfp-36, which is highly expressed in response to growth factors and in several hematopoietic cell lines. In the present studies, we investigated the possibility that TTP is phosphorylated in intact cells. In NIH/3T3 cells that were made to overexpress TTP constitutively, we found that the protein was phosphorylated on serine residues, and that this phosphorylation was rapidly (within 10 min) stimulated by several mitogens. In cell-free assays, recombinant mouse TTP was a substrate for the mitogen-activated protein (MAP) kinase. By a combination of protease digestion experiments and site-directed mutagenesis strategies, we found that serine 220 was phosphorylated by p42 MAP kinase in vitro. Expression of mutant TTP in fibroblasts confirmed that serine 220 was one of the major, mitogen-stimulated phosphorylation sites on the protein in intact cells. These results suggest that TTP may be phosphorylated by MAP kinases in vivo and that this phosphorylation may regulate its function.


INTRODUCTION

Mouse tristetraprolin (TTP)() is a basic, proline-rich protein of M 33,600 that contains three PPPPG repeats (1) . Also known as Nup475 (2) and TIS11 (3), TTP contains two putative CCCH zinc fingers, and the recombinant protein has been shown to bind Zn(2) . Recently, several other proteins have been recognized that contain similar types of putative zinc finger structures, including those encoded by the mammalian cDNAs TIS11b (cMG1) (3, 4) and TIS11d (3) , those encoded by the Drosophila genes unkempt(5) and DTIS11(6) , and those encoded by the yeast genes CTH1 and CTH2.() Although TTP has been localized to the cell nucleus in mouse fibroblasts (2) and may well be a transcription factor, neither TTP nor any of the related zinc finger proteins has been shown to bind to specific DNA target sequences.

Expression of the gene that encodes TTP, Zfp-36(7) , is induced by several mitogens including serum, insulin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and phorbol 12-myristate 13-acetate (PMA) (1, 2, 3) . In quiescent mouse fibroblasts, TTP mRNA is virtually undetectable, but it accumulates within 10 min of mitogen stimulation, peaks at 30 min, and returns to basal levels within 2 h. This is due primarily to increased transcription, and the resulting mRNA has a half-life of 15-30 min (1, 8) . In the mouse, TTP mRNA is highly expressed in lung, intestine, lymph node, spleen, and thymus, with lower expression in liver and kidney, and negligible expression in brain (1, 2) . In addition, the mRNA is constitutively highly expressed in several hematopoietic cells, with very high expression in macrophages and neutrophils, and somewhat lower expression in B and T lymphocytes.() Recently, targeted disruption of Zfp-36 in mouse embryonic stem cells has been used to create mice that do not express TTP.() These mice are normal at birth, but soon become runted and cachectic, develop patchy alopecia and dermatitis, and often die within the first few months after birth. In addition, the mice have a marked increase in granulocytes in the bone marrow, spleen, lymph nodes, and peripheral blood, and a decrease in B and T lymphocytes in the spleen and peripheral blood. This phenotype suggests that TTP could play a role in granulopoiesis or in the development of other hematopoietic cell lineages.

In the present study, we demonstrate that TTP is a phosphoprotein, and we show that its phosphorylation on serine residues is stimulated by several mitogens including serum, PDGF, FGF, and PMA, but not by dibutyryl cAMP or forskolin. Furthermore, we show that serine 220 is one of the residues that is phosphorylated by p42in vitro and that this site is also phosphorylated in intact cells. These experiments suggest that, in addition to controlling the availability of TTP protein by regulating gene transcription, certain mitogens may also control its function by regulating its reversible phosphorylation.


EXPERIMENTAL PROCEDURES

Plasmid Subcloning

pmMT-neo was created by replacing the 4.3-kb EcoRI-SalI human -actin gene promoter fragment in pHApr-1-neo (9) with the 0.7-kb EcoRI-BglII mouse metallothionein gene fragment from pmMT-1 (10, 11) . pmMT-TTP was then created by ligating a 1.1 HindIII-BglII mouse TTP cDNA fragment from clone DU2 (1) , which contains the entire TTP coding region, into the HindIII, BamHI site of pmMT-neo.

The plasmid encoding GST/TTP, pGEX-TTP, was created by ligating the 1.1-kb NcoI-BglII fragment of DU2 into the SmaI site of pGEX-2T. The plasmids encoding pGEX-TTP(163-215) and pGEX-TTP(163-235) were created by first using the polymerase chain reaction to create the appropriate DU2 (TTP cDNA) fragments with HindIII and XbaI sites added at the 5` and 3` ends of each fragment, respectively. DNA primers 5`-GGCCTCTAGACCTAGCTCTCCCTGGCCAGCCC-3` and 5`-CGATAAGCTTTTACCCAGGGGGCGGTGGGGAGCT-3` were used to amplify the region encoding amino acids 163-215; DNA primers 5`-GGCCTCTAGACCTAGCTCTCCCTGGCCAGCCC-3` and 5`-CGATAAGCTTTTATCTTCGAGTCACAGGGGTCCC-3` were used to amplify the region encoding amino acids 163-235. Next, the amplified DNA fragments were subcloned into the HindIII, XbaI site of pGEX-KG (12) . pGEX-TTP(S220A) was created by first changing the codon that encodes S220 in DU2 from TCC to GCC by the polymerase chain reaction primer overlap mutagenesis technique (13) . Next, the 1.7-kb NcoI-EcoRV fragment was cleaved from the mutagenized TTP cDNA plasmid and ligated into the NcoI, HindIII site of pGEX-KG. Similarly, pmMT-TTP(S220A) was created by ligating a 1.1-kb BamHI-BglII fragment from the mutagenized TTP plasmid into the BamHI site of pmMT-neo.

Cells and Culture

NIH/3T3 cells (ATCC CRL 1658, American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Hazleton Research Products, St. Lenexa, KS) supplemented with 10% fetal bovine serum (FCS) (Life Technologies, Inc.). The TTP-deficient fibroblasts were prepared by first isolating primary embryonic fibroblasts from 14-17-day C57BL/129Sv mouse embryos (14); these embryos were the result of gene targeting experiments in mouse embryonic stem cells in which a Neo gene had been inserted into the protein coding region of the gene that encodes TTP. The primary fibroblasts cultures were then immortalized by subjecting them to the 3T3 selective procedure of Todaro and Green (15) . The immortalized fibroblasts expressed neither detectable TTP mRNA as assessed by Northern blotting nor detectable TTP protein as assessed by immunoprecipitation of [S]cysteine-labeled protein. The TTP-deficient fibroblasts were maintained in DMEM supplemented with 10% FCS.

Transfection

To establish stable cell lines, plasmids were introduced into NIH/3T3 cells by CaPO transfection. 24 h before transfection, cells were plated at 0.5 10 cells/100-mm plate, and 2 h before transfection, fresh medium was added to the plates. A solution was prepared by mixing HO, 10 µg of plasmid DNA, and 125 µl of 1 M CaCl in that order for a total volume of 0.5 ml, and this solution was added dropwise to 0.5 ml of 2 HBS (28 mM NaCl, 1.5 mM NaPO, 50 mM HEPES, pH 6.95) through which N was bubbling. 30 min later, 1 ml of DNA solution was added to each plate of cells. Following a 12-16-h incubation, the cells were washed three times with TBS (137 mM NaCl, 5 mM KCl, 25 mM Tris, pH 7.4), and then fresh medium was added. Neo cells were selected with medium supplemented with 0.3 mg/ml Geneticin (G418 sulfate) (Life Technologies, Inc.) for 2-3 weeks.

In Vitro Transcription and Translation

DU2 was linearized with XbaI and transcribed with T7 RNA polymerase (Life Technologies, Inc.) to obtain sense mRNA. In vitro translation was performed in a rabbit reticulocyte translation system (Promega, Madison, WI) with 100 µCi of [S]cysteine/reaction, incubating 1 h at 30 °C. Small aliquots of the reaction mixture were phosphorylated with p42 as described below.

In Vitro Phosphorylation and Proteolytic Cleavage

pGEX-TTP plasmids were transformed into Escherichia coli strain BL21/DE3, and the GST fusion proteins were purified from these cells with glutathione-Sepharose (Pharmacia) as described previously (16) . Aliquots of the GST fusion protein preparations were analyzed on 9% denaturing polyacrylamide gels that were stained with Coomassie Blue (Diversified Biotech, Scientific Technologies, Raleigh, NC). The GST fusion proteins were incubated with activated p42 for 30 min at 30 °C in 10 mM MgCl, 0.1 mM ATP, 1 mM dithiothreitol, 0.1 mM EGTA, 0.1 mM NaVO, and 25 mM Tris, pH 7.2. The purified, activated recombinant p42 was generously provided by Dr. Paul Dent and Dr. Tom Sturgill, University of Virginia (17) . The reaction products were separated on a 9% denaturing polyacrylamide gel, and the gel was transferred to nitrocellulose with a TEX50 wet transfer apparatus (Hoeffer, San Francisco, CA) using 25 mM Tris, 480 mM glycine, 0.1% SDS, and 50% methanol transfer buffer. Because small amounts of less than full-length fusion proteins were present in the GST/TTP protein preparations, only the band shown by autoradiography to contain the full-length GST/TTP fusion protein was excised. The nitrocellulose piece was incubated for 16 h at 37 °C in 50% pyridine, 50% ammonium acetate, pH 8.9, to elute the protein (18) , and the protein was precipitated by the addition of 20% trichloroacetic acid and 50 µg of carrier -globulin (Sigma). The protein pellet was resuspended and proteolytically digested with trypsin (L-(tosylamido 2-phenyl)ethyl chloromethyl ketone-treated, Worthington, Freehold, NJ) in 0.05 M NHHCO, CHCN (95:1), with endoproteinase Arg-C (Sigma) in 100 mM Tris, pH 8.5, with endoproteinase Glu-C (Sigma) in 100 mM Tris, pH 7.8, or with endoproteinase Asp-N (Sigma) in 100 mM Tris, pH 8.5. The products of these reactions were separated on 20% denaturing polyacrylamide gels.

Antibody Preparation

The peptide MDLSAIYESLQSMSHDLSSDHGGC, which corresponds to the 24 NH-terminal amino acids of mouse TTP, was synthesized and purified as described (19) . Several rabbit antisera were raised against this peptide coupled to keyhole limpet hemacyanin by Immuno-Dynamics, Inc. (La Jolla, CA). Immunospecific antibody was purified using the immunogenic peptide and a membrane affinity chromatography capsule (no. 11016, Amicon, Beverly, MA) as described by the manufacturer.

Cell Labeling

Confluent 60-mm plates of cells were serum-deprived by washing three times with DMEM and then incubating them with DMEM supplemented with 1% (w/v) bovine serum albumin (BSA) (Life Technologies, Inc.) for 24 h. When appropriate, the metallothionein promoter was induced by adding sterile ZnSO solution to the plates at a final concentration of 100 µM for the final 12-15 h of the serum deprivation period. For cells labeled with [S]cysteine, the cells were first washed three times with cysteine-free DMEM (Biofluids, Rockville, MD) and then incubated with 2 ml of cysteine-free DMEM supplemented with 1% BSA and 100 µM ZnSO for 30 min. Next, 0.5 mCi of [S]cysteine (DuPont NEN) was added directly to each dish and incubated for 2 h followed by other treatments as described under ``Results.'' For cells labeled with P, the cells were first washed three times with Krebs buffer (65 mM NaCl, 4.7 mM KCl, 1.4 mM CaCl, 1.2 mM MgSO, 25 mM NaHCO, 25 mM glucose, and 10 mM HEPES, pH 7.4) and then incubated in 2 ml of Krebs buffer supplemented with 0.3 mCi of [P]HPO (DuPont), 1% BSA, and 100 µM ZnSO, followed by other treatments as described under ``Results.''

Immunoprecipitation

Total cellular protein lysates were prepared by washing labeled cells three times with ice-cold Krebs buffer and scraping them with a rubber policeman into 0.5 ml of ice-cold Nonidet P-40 lysis buffer (1% (w/v) Nonidet P-40, 5 mM EDTA, 0.15 M NaCl, 50 mM Tris, pH 8.3). Following sonication for 15 s in a W-380 cup horn sonicator (Heat Systems, Farmingdale, NY), the lysates were cleared by centrifugation in microfuge tubes in a TL100 table-top ultracentrifuge (Beckman, Fullerton, CA) at 88,000 g for 20 min at 4 °C. The samples were matched for trichloroacetic acid-precipitated radioactive counts and were then incubated with normal rabbit serum (1:100 dilution) for 1 h and 6 mg of protein A-Sepharose (Pharmacia) for an additional 1 h. Following a 2-min centrifugation at 500 revolutions/min in a microfuge 11 (Beckman) to pellet the protein A-Sepharose, the supernatant was transferred to a new tube and incubated for about 12 h with 3.3 µg of TTP antibody and for an additional 1 h with 6 mg of protein A-Sepharose. Following four washes with wash buffer (0.5% Nonidet P-40, 1 mM EDTA, 0.15 M NaCl, 50 mM Tris, pH 8.3), 100 µl of an SDS buffer (30% sucrose, 0.45 M dithiothreitol, 0.06 M EDTA, 6% SDS, 0.06 mg/ml pyronin y, diluted 1:4 with phosphate-buffered saline) was added to the resin; the suspension was mixed vigorously, boiled for 5 min, and the resin was pelleted by centrifugation for 5 min at 1500 revolutions/min in a microfuge. Immunoprecipitated proteins were separated by 9% denaturing SDS-polyacrylamide gel electrophoresis.

Cloning of a Bovine TTP cDNA

The bovine TTP cDNA was cloned() by screening a bovine aortic endothelial cell cDNA library (Stratagene, La Jolla, CA) with a human TTP cDNA probe (7) . Cloning and sequencing methods were as described previously (1) .


RESULTS

Expression of TTP in NIH/3T3 Cells

NIH/3T3 cells were serum deprived for 24 h and exposed to [S]cysteine for 4 h and to 20% (v/v) FCS for varying times during those 4 h. Total cellular protein lysates were prepared, and protein was immunoprecipitated from the lysates with a polyclonal antibody that had been raised against a peptide corresponding to the NH-terminal 24 amino acids of TTP (Fig. 1). A protein species was precipitated that migrated as a broad band with an apparent molecular mass of about 43 kDa. This protein was not detectable in quiescent NIH/3T3 cells but accumulated within 1 h of FCS exposure, peaked at about 2 h, and was decreased but still present at 4 h. Although TTP has a predicted molecular mass of 33.6 kDa, several observations indicate that the observed 43-kDa protein is TTP. First, the time course of expression of the protein following FCS stimulation lagged slightly behind that of the TTP mRNA which is present in these cells within 10 min of serum exposure, peaks at 30-45 min, and returns to near basal levels within 2 h (1) . Second, no protein of this M was precipitated by the preimmune serum (data not shown). Third, precipitation of the protein could be competed completely by the addition of the immunogenic peptide to the lysates prior to immunoprecipitation with the TTP NH-terminal antibody (not shown). Fourth, when a similar experiment was performed using a polyclonal antibody that had been raised against a peptide corresponding to the COOH-terminal 20 amino acids of TTP, a protein was precipitated that had the same apparent electrophoretic mobility and the same time course of expression (not shown). In this experiment, the corresponding immunogenic peptide also blocked precipitation of the protein with the TTP COOH-terminal antibody (not shown). Finally, the 43 kDa species was not precipitated by the TTP NH-terminal antibody from lysates isolated from serum-stimulated fibroblasts prepared from TTP-deficient mice (see below). Taken together, these experiments indicate that the TTP NH-terminal antibody was able to precipitate TTP from cell lysates; the remainder of the immunoprecipitation experiments that are described in this paper were performed using this antibody.


Figure 1: Time course of FCS-stimulated TTP accumulation in NIH/3T3 cells. Confluent, serum-deprived cells were exposed to [S]cysteine for a total of 4 h and to 20% FCS for the indicated times. Cell lysates were prepared and used for immunoprecipitation with the TTP NH-terminal antibody. The precipitated protein was separated on a 9% SDS-polyacrylamide gel. See the text for further details.



Because almost no TTP is detectable in serum-deprived cells, we established cell lines that constitutively express TTP, even in the serum-deprived state, by stably transfecting NIH/3T3 cells with a plasmid containing the TTP cDNA linked to the metallothionein (mMT) promoter. In this plasmid, the 3`-untranslated region of the TTP cDNA, which contains several AU-rich sequences that are known to destabilize the TTP mRNA, had been removed and replaced with an SV40 polyadenylation signal. The plasmid also contained a neo gene; consequently, neo cell clones were isolated following transfection, and three of these cell lines were assayed for expression of exogenous TTP by immunoprecipitation of [S]cysteine-labeled protein (Fig. 2). Line A did not express exogenous TTP, but only expressed endogenous TTP following a 2-h exposure to FCS. However, both lines B and C expressed TTP in the quiescent state, and interestingly, there was a shift in electrophoretic mobility of the protein following a 2-h FCS exposure. This suggested that FCS caused a covalent modification of TTP of some type, possibly phosphorylation.


Figure 2: Effect of FCS on TTP protein expression in mMT/TTP-expressing NIH/3T3 cells. Confluent NIH/3T3 (3T3) cells or NIH/3T3 cells that stably expressed mMT/TTP (A-C) were serum-deprived for 24 h; 100 µM ZnSO was added for the final 15 h. The cells were then incubated with [S]cysteine for 2 h followed by an additional 2 h of incubation under control conditions or with 20% FCS. Cell lysates were prepared, protein was precipitated with the TTP NH-terminal antibody, and the precipitated protein was separated on a 9% SDS-polyacrylamide gel. See the text for further details.



Phosphorylation of TTP

To determine if FCS stimulated phosphorylation of TTP, mMT/TTP-expressing cells (line B) were exposed to P for 4 h and to FCS for varying times during those 4 h, and an immunoprecipitation was performed of lysates from those cells (Fig. 3). In quiescent cells, two protein species were precipitated that were each about 43 kDa. The upper species was detectable in untransfected, serum-deprived NIH/3T3 cells as well as in TTP-deficient fibroblasts that had been exposed to similar conditions (Fig. 3); these results indicate that the upper band is not TTP, but rather, a co-immunoprecipitating protein that probably contains a shared epitope. However, the lower species was not detectable in untransfected, serum-deprived NIH/3T3 cells, or in TTP-deficient fibroblasts, under similar conditions (Fig. 3), suggesting that it is basally phosphorylated, exogenous TTP. Within 10 min of FCS stimulation, the lower species was no longer detectable, and there was a marked increase in the intensity of the upper species; this effect was constant to at least 2 h ( Fig. 3 and data not shown). Because the effect was not seen in immunoprecipitated protein from FCS-stimulated, untransfected NIH/3T3 cells (Fig. 3), FCS probably caused an increase in TTP phosphorylation and a change in TTP electrophoretic mobility so that it then comigrated with the epitope-related upper band. Therefore, it can be concluded that although TTP is phosphorylated in quiescent cells that constitutively express it, FCS causes a substantial increase in TTP phosphorylation and a corresponding shift in its electrophoretic mobility, similar to the shift seen with [S]cysteine-labeled TTP in NIH/3T3 cells following FCS stimulation (Fig. 2).


Figure 3: FCS-induced TTP phosphorylation in NIH/3T3 cells, TTP-deficient fibroblasts (TTP(-/-)), and mMT/TTP-expressing cells. Confluent cells were serum-deprived for 24 h; 100 µM ZnSO was added for the final 15 h. The cells were then incubated with P for 2 h followed by an additional 15 min of incubation with 20% FCS. Cell lysates were prepared, protein was precipitated with the TTP NH-terminal antibody, and the protein was separated on a 9% SDS-polyacrylamide gel. Other details are as described in the text.



To determine if other mitogens could cause a similar increase in TTP phosphorylation, mMT/TTP-expressing cells were labeled with P and exposed to several mitogens for 15 min, and TTP from these cells was immunoprecipitated (Fig. 4). FCS, PMA, PDGF, and FGF all caused a substantial increase in TTP phosphorylation, but dibutyryl cAMP and forskolin had no detectable effect; these studies suggested that the cyclic AMP-dependent protein kinase probably did not mediate TTP phosphorylation in cells under these conditions.


Figure 4: Effect of mitogens on TTP phosphorylation in mMT/TTP-expressing NIH/3T3 cells. Confluent cells were treated as described in the legend to Fig. 3. Following a 2-h exposure to P, the cells were exposed to control conditions, 1% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) FCS, 1.6 µM PMA, 10 ng/ml PDGF, 10 nM FGF, 1 mM dibutyryl cAMP, or 100 µM forskolin for 15 min. Immunoprecipitation was then carried out as described in the legend to Fig. 3.



To determine which amino acids on TTP are phosphorylated in vivo, the intensely phosphorylated upper band from FCS-stimulated mMT/TTP-expressing cells, and both the upper and lower bands from quiescent mMT/TTP-expressing cells, were each isolated and subjected to phosphoamino acid analysis. In each case, only phosphoserine was detected (data not shown).

TTP Phosphorylation by p42in Vitro

Many signaling cascades that are initiated by extracellular signals at cell surface receptors converge on a common signaling pathway that results in the activation of mitogen-activated protein (MAP) kinases (also known as extracellular signal-regulated kinases, or ERKs) (see Refs. 20 and 21 for reviews). Among the many substrates that have been identified for the MAP kinases are several transcription factors including c-Jun (22) , c-Fos (23) , c-Myc (24, 25) , and p62(26) . To determine whether TTP was also a MAP kinase substrate, TTP was synthesized in a rabbit reticulocyte lysate system, and the S-labeled TTP was incubated with one of the activated MAP kinase isoforms, p42(17) (Fig. 5). The electrophoretic mobility of TTP following incubation with p42 was shifted relative to protein that had been incubated under control conditions (Fig. 5), indicating that p42 can phosphorylate TTP in vitro. This electrophoretic shift was similar to that seen with TTP isolated from FCS-stimulated NIH/3T3 cells (Fig. 2). To identify the site(s) in TTP that were phosphorylated by p42, a GST/TTP fusion protein was created and used as a p42 substrate. Following incubation of GST/TTP with p42 in the presence of [P]ATP, GST/TTP-P was isolated and exhaustively digested with several proteases (Fig. 6). One of these proteases, endoproteinase Asp-N, cleaved the protein to produce one major phosphopeptide of about 6 kDa. Among the TTP fragments that are predicted to be created following endoproteinase Asp-N digestion are two fragments that are about 6 kDa (Fig. 7). Consequently, two other fusion proteins were created that fused GST with these two TTP fragments, amino acids 163-215 and amino acids 163-235, and each fusion protein was incubated with p42 (Fig. 8). Although GST/TTP(163-235) was a substrate for p42 under these conditions, GST/TTP(163-215) was not, indicating that the phosphorylation site(s) was (were) between amino acids 216 and 235. In this region of TTP, there are 3 serines (1) , but only serine 220 falls within the MAP kinase phosphorylation consensus sequence of P-X-S/T-P (20) . To determine if serine 220 was phosphorylated by p42, another fusion protein was created that fused GST with a full-length TTP in which serine 220 had been mutated to alanine. This fusion protein, GST/TTP(S220A), was phosphorylated by p42 to a much lesser extent than was GST/TTP under identical conditions (Fig. 8). Consistent with this result, a synthetic peptide corresponding to TTP amino acids 211-230 was a good substrate for p42in vitro, whereas an otherwise identical peptide in which alanine was substituted for serine 220 was a much poorer substrate (Fig. 9). Preliminary kinetic analysis indicated that phosphorylation of the peptide that corresponded to amino acids 211-230 was half-maximal at about 250 µM substrate (not shown). These experiments demonstrate that serine 220 is a major phosphorylation site for p42in vitro.


Figure 5: Effect of p42 on in vitro synthesized TTP. TTP was synthesized in a rabbit reticulocyte lysate system and then incubated in a kinase buffer containing ATP and no kinase (C) or p42 (MAP) for 30 min. The incubated protein was separated on a 9% SDS-polyacrylamide gel. The experiment was carried out in duplicate with five times more protein in the second set of samples.




Figure 6: Proteolytic digestion of GST/TTP that had been phosphorylated by p42. GST/TTP was phosphorylated by p42in vitro as described in the text, and the reaction mixture was separated on a 9% denaturing polyacrylamide gel. Full-length GST/TTP-P was excised from the dried gel, and the protein was eluted from the gel slice. The protein was then incubated with nothing (control), trypsin, endoproteinase Arg-C, endoproteinase Asp-N, or endoproteinase Glu-C. The digested protein was separated on a 20% SDS-polyacrylamide gel. Other details are as described in the text.




Figure 7: Predicted TTP endoproteinase Asp-N proteolytic fragments. The major TTP proteolytic fragments following digestion with endoproteinase Asp-N are displayed. Diagrams of two GST fusion proteins that were constructed based on the size of the digested fragments are also shown.




Figure 8: Phosphorylation of GST/TTP fusion proteins by p42in vitro. A, the indicated fusion proteins were incubated with p42 in the presence of [-P]ATP for 30 min and then separated on a 9% SDS-polyacrylamide gel. An autoradiograph of this gel is shown. B, equal amounts of the total protein from the indicated GST fusion protein preparations were separated on a 9% SDS-polyacrylamide gel that was then stained with Coomassie Blue. This gel is identical to that from which the autoradiograph in A was prepared.




Figure 9: Peptide phosphorylation by p42. Synthetic peptides corresponding to the indicated amino acids of the mouse TTP protein were incubated with p42 for 30 min under the conditions described in the text. Following incubation, the peptides were separated from unincorporated [-P]ATP on a 20% SDS-polyacrylamide gel; an autoradiograph of the gel is shown. Exposure time was 1.3 h.



Phosphorylation of Serine 220 in Vivo

To determine if serine 220 was phosphorylated in intact cells in response to mitogen stimulation, two stable NIH/3T3 cell lines were created that each constitutively expressed TTP(S220A) under control of the mouse metallothionein promoter. Phosphorylation of TTP(S220A) in these cells was compared to that of wild-type TTP in mMT/TTP-expressing cells (Fig. 10). Following FCS stimulation, an increase in phosphorylation of TTP(S220A) was seen, as was a shift in the electrophoretic mobility of the mutant protein. However, the mobility shift of TTP(S220A) was significantly decreased compared to the mobility shift of wild-type TTP. The same effect on electrophoretic mobility was seen in protein precipitated from both [S]cysteine and P-labeled cells (Fig. 10). The fact that the mobility shift of TTP(S220A) was decreased indicates that serine 220 is a site of FCS-stimulated TTP phosphorylation, but the fact that some of the TTP(S220A) mobility shift remained suggests that there are other phosphorylation site(s) that remain to be identified. The extent of the electrophoretic shift seen with the mutant protein appeared to be about 50% of that seen in the wild-type protein, suggesting the possibility that only one FCS-stimulated phosphorylation site remains to be identified in TTP(S220A).


Figure 10: The effect of FCS on TTP and TTP(S220A) phosphorylation in NIH/3T3 cells. Cells that stably expressed mMT/TTP or mMT/TTP(S220A) were serum-deprived for 24 h; 100 µM ZnSO was added for the final 15 h. The cells were then incubated (A) with [S]cysteine for 2.5 h or (B) with P for 2 h, followed by an additional 15 min incubation under control conditions or with 20% FCS. Immunoprecipitation, electrophoresis, and autoradiography were carried out as described in the legend to Fig. 3. Note that mMT/TTP(S220A) A cells expressed roughly comparable amounts of S-labeled TTP compared to wild-type mMT/TTP cells.




DISCUSSION

These experiments have demonstrated that TTP is a serine phosphoprotein and that its phosphorylation in fibroblasts is stimulated by several mitogens, including FCS, PMA, PDGF, and FGF, but not by forskolin or dibutyryl cAMP. Phosphorylation of at least 2 serine residues on TTP is enhanced following FCS stimulation, and we have identified serine 220 as 1 of those residues. In addition, we have shown that serine 220 is phosphorylated in cell-free assays by one of the MAP kinase isoforms, p42. These results suggest that TTP function might be regulated by mitogens at the level of reversible phosphorylation.

Since no function has been ascribed to TTP, as a transcription factor or otherwise, we cannot determine whether or not this covalent modification is functionally significant. However, the fact that the M of the protein is shifted significantly by mitogen stimulation in cells suggests that this modification occurs in vivo with significant stoichiometry; in fact, the lower TTP species present in the basal or serum-deprived cells completely disappears in favor of the more highly phosphorylated species after only 10 min of mitogen treatment. In cell-free kinase assays, the GST-TTP fusion protein can be phosphorylated by p42. Finally, serine 220 and its surrounding minimal MAP kinase consensus sequences are conserved in the mouse (1, 2, 3) , human (7) , and bovine sequences; all of these species contain the sequence L-S-P at this site (Fig. 11). These data suggest that there are at least two physiologically significant phosphorylation sites on TTP, one being serine 220, and that phosphorylation on one or more sites occurs with a stoichiometry to make it physiologically important.


Figure 11: Alignment of the mouse (mttp), rat (rttp), bovine (bttp), and human (http) TTP protein sequences. Alignments were performed by the program Pileup (Genetics Computer Group, Madison, WI); periods in the sequences indicate spaces introduced by the program to optimize alignments. Sequence identities are indicated by asterisks (*). The putative CCCH zinc finger regions have been placed in brackets, and the C and H residues that may coordinate Zn have been highlighted. The identified MAP kinase site in the mouse protein (Ser) is noted with a large arrow, and the remaining seven minimal MAP kinase consensus sequences (S-P) that are conserved in all four proteins are noted with small arrows.



Although our work shows that p42 can phosphorylate TTP in cell-free assays and suggests that p42 might also phosphorylate TTP in intact cells, it is possible that other kinases phosphorylate TTP in intact cells rather than, or in addition to, p42. Because serine 220 in TTP is followed by a proline, it seems likely that these additional kinase(s) are proline-directed kinases. In addition to p42, other proline-directed kinases include two other MAP kinases, p44(20, 21) and p54(27) , and a group of MAPK-related kinases, the c-Jun amino-terminal kinases (JNKs) (28) . Each of these kinases has a similar substrate specificity (20, 28) and could conceivably phosphorylate TTP serine 220, as well as other serines in TTP. Another proline-directed kinase is the cdc2 kinase (29). However, none of the serines in TTP fall within the cdc2 phosphorylation consensus sequence, S-P-X-R/K (29) . Finally, most of the mitogens that increase TTP phosphorylation in cells can activate protein kinase C; however, the GST-TTP fusion protein was not a substrate for the constitutively active form of mixed brain protein kinase C isozymes in cell-free assays.

The facts that TTP is encoded by an early response gene, that it contains two putative zinc fingers, and that it is a nuclear protein, all suggest (but do not prove) that it is a transcription factor. MAP kinases are known to phosphorylate several other transcription factors, and this alters function of these proteins in several ways (20) . In some cases, MAP kinase phosphorylation alters DNA binding. For example, when ATF-2 that has been isolated from cells is treated with protein phosphatase 2A, ATF-2-binding to the DNA cAMP response element is markedly reduced (30) . However, in vitro phosphorylation of ATF-2 by MAP kinase can restore DNA binding activity (30) . Conversely, in the case of c-Jun, the MAP kinases p42 and p44 can phosphorylate a site within the c-Jun COOH-terminal domain, and phosphorylation at this site inhibits DNA binding (22) . It is not obvious how phosphorylation of TTP at serine 220 would alter DNA binding, since this residue lies well outside of the putative zinc finger region, which is between amino acids 95 to 159 in the mouse protein (Fig. 11).

MAP kinase phosphorylation of several other transcription factors is thought to alter the trans-activating ability of these proteins. For example, serine 62 in c-Myc is a major phosphorylation site in vivo and also a phosphorylation site for MAP kinase in vitro(24, 25) . Phosphorylation of c-Myc at serine 62 has been associated with increased trans-activation of gene expression (24, 25) . Similarly, the trans-activating ability of the basic-leucine zipper transcription factor NF-IL6 is also increased by MAP kinase phosphorylation, in this case, on threonine 235 (31). It is conceivable that MAP kinase phosphorylation might alter the function of TTP in a similar way; however, the putative trans-activating domains of TTP have not yet been identified, so this possibility cannot be tested at this point.

Other potential functional changes induced by TTP phosphorylation could include effects on nuclear-cytoplasmic partitioning and/or protein-protein interactions. For example, the yeast transcription factor SWI5 is localized to the nucleus in G where it activates transcription of the HO endonuclease; during other phases of the cell cycle, SWI5 translocates to the cytoplasm (32) . Phosphorylation of SWI5 is catalyzed by the Cdc28 kinase in conjunction with an activating cyclin subunit and appears to be responsible for the nuclear to cytoplasmic translocation (33) . Phosphorylation of TTP could also affect its association with putative regulatory proteins, as in the NF-B/I-B interaction (34, 35) .

To determine exactly how phosphorylation regulates TTP function, it will be important to determine the remaining serine phosphorylation site(s). However, although serine 220 was clearly the dominant phosphorylation site in our cell-free kinase assays using p42, phosphorylation on other serine(s) was relatively minor in this system; hence, the other phosphorylation sites may be difficult to identify using the same approach. ()In addition, efforts to identify the other sites will be complicated by the fact that the mouse TTP protein contains 55 serine residues; 8 of these fall into the minimal MAP kinase recognition sequence of S-P (1, 20, 21) and are conserved among the mouse, rat, human, and bovine TTP proteins (Fig. 11). Efforts are currently underway to identify these remaining site(s).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by the Howard Hughes Medical Institute Laboratory Graduate Student's Support Fund.

Supported by National Institutes of Health Training Grant K11-DK02227-02.

**
Associate of the Howard Hughes Medical Institute.

§§
Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: P. O. Box 3897, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-8760; Fax: 919-684-5458.

The abbreviations used are: TTP, tristetraprolin; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; PMA, phorbol 12-myristate 13-acetate; dibutyryl cAMP, dibutyryl cyclic adenosine monophosphate; MAP kinase, mitogen-activated protein kinase; mMT, mouse metallothionein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GST, glutathione S-transferase; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase.

M. J. Thompson, W. S. Lai, G. A. Taylor, and P. J. Blackshear, submitted for publication.

G. A. Taylor and P. J. Blackshear, unpublished results.

G. A. Taylor, D. M. Lee, W. S. Lai, M. J. Thompson, B. F. Haynes, and P. J. Blackshear, manuscript in preparation.

W. S. Lai, M. J. Thompson, Y. I. Liu, and P. J. Blackshear, manuscript in preparation.

Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Dr., Madison, WI 53711.


ACKNOWLEDGEMENTS

We are very grateful to Drs. Paul Dent and Tom Sturgill for the purified p42 MAP kinase.


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