INTRODUCTION

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Activating transcription factor 2 (ATF2)¹ is a member of the ATF/CREB family of basic region leucine zipper (bZIP) DNA-binding proteins that regulates transcription by binding to a conserved CRE elements in the promoter of genes (1, 2). We have shown that stimulation of T or B lymphocytes via the antigen receptor is associated with a marked induction of ATF2 binding to consensus CRE elements as well as to CRE elements in the promoter of proliferating cell nuclear antigen at late stages of activation or differentiation (3). In addition, induction of CRE binding activity was observed by us and others in cloned T cells stimulated with IL-2 (4) and in human peripheral blood T cells stimulated by PHA (5). Conversely, inhibition of T cell proliferation by rapamycin, a potent macrolide immunosuppressant, was associated with a marked inhibition of ATF2 binding activity (3, 6). This indicates that transactivation of ATF2 is tightly regulated and may play an important role during lymphocyte activation.

The transcription factor ATF2 has been implicated in the induction of cytokine gene expression after engagement of the antigen receptor in lymphocytes (7, 8). Specifically, ATF2 was implicated in the transcription of tumor necrosis factor  (7), in tumor growth factor  (8), and in the virally induced interferon  (9). An important role for ATF2 has also been demonstrated in the expression of T cell-specific genes (10-13). ATF2 binds to the conserved CRE decamer motif in the CD8 promoter (10) and in TCR V promoter (11). Isoforms of the CRE-BP1 DNA (ATF2) were found to mediate activity of a T cell-specific enhancer required for expression of the CD3 gene of the T cell receptor complex (12). Because this motif is also present in the enhancer and promoter of the TCR  and  genes, it has been suggested that ATF2 may mediate expression of other members of the CD3/TCR complex during T cell activation and differentiation (12).

ATF2 was also implicated in the pathogenesis of HTLV-1 (14, 15), the etiologic agent of adult T cell leukemia, an aggressive malignancy of helper T lymphocytes in humans. Specifically, ATF2 was shown to bind and transcriptionally activate the virus-encoded transactivator protein, Tax, leading to induction of HTLV-1 gene expression (9-12). Thus, it appears that pathological processes by oncogenic virus expression in human T lymphocytes utilize cellular processes that up-regulate ATF2 transcription factors during T cell activation.

Evidence indicated that full-length ATF2 is inactive due to intramolecular inhibition exerted by its DNA binding domain (16). This led to the hypothesis that ATF2 interaction with other proteins releases this intramolecular inhibition and transactivates the protein (16). Indeed, ATF2 transcriptional activation was shown to involve interaction with several different proteins including: E1A (17-19), the tumor suppressive gene product Rb (20), the high mobility group HMG (21), NF-B (21), and c-Jun (22). Therefore, the function of ATF2 may be determined by its specific interaction with other inducer proteins. Transcriptional activation of ATF2 was shown to also be induced by phosphorylation mediated by stress-activated protein kinases including Jun N-terminal kinase (JNK), a subgroup of the MAP kinase family (23-26). Furthermore, studies have shown that cell stimulation via pro-inflammatory cytokines or ultraviolet radiation is associated with rapid phosphorylation of ATF2 by JNK, implicating a role for ATF2 in rapid signaling processes via JNK transduction pathways (24). JNK has been implicated in integration of different signaling pathways leading to T cell activation via the TCR/CD3 (27). Taken collectively, this evidence leads to the possibility that transcriptional activation of ATF2 may be tightly regulated by early and late signaling processes during T cell activation.

To elucidate the mechanisms that regulate ATF2 in T cell activation, we searched for proteins that interact with ATF2 in vivo. The genetic approach of the yeast two-hybrid was employed using the full-length human ATF2 cDNA used as a "bait." The search was performed using a cDNA library of PHA-activated human peripheral blood leukocytes. We report here the isolation and characterization of a cDNA clone that encodes a new member of the ubiquitin-conjugating enzyme family, which specifically interacts with ATF2 in the yeast two-hybrid system and in vitro in Far Western analysis. An identical sequence was recently reported as the human homolog of the yeast ubiquitin-conjugating enzyme 9, hUBC9 (28-30). We demonstrate that hUBC9 protein localizes predominantly in the nuclei of control and stimulated T cells. hUBC9 belong to a family of ubiquitin-conjugating enzymes (E2) that participate in the linking of C-terminal glycine residues of ubiquitin to specific lysine residues of target proteins (for review, see Refs. 31-33). This ubiquitination leads to target protein degradation by the 26 S proteasome. We demonstrate that ATF2 is ubiquitinated in vivo and in vitro and provide evidence that hUBC9 facilitates the ubiquitination of ATF2 in vitro. We further demonstrate that changes in abundance of degradation products of ATF2 occur rapidly upon T cell activation concomitant with induction phosphorylation of ATF2. These results taken collectively implicate the proteasome pathway and specifically hUBC9 in the regulation of ATF2 during T cell activation.

	MATERIALS AND METHODS

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Reagents-- Affinity-purified antibody specific for conjugated ubiquitin was a kind gift of Dr. Arthur L. Haas (Medical College of Wisconsin, Milwaukee, WI) and Dr. C. Pickart (Johns Hopkins University, Baltimore, MD). Anti-ATF2 polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY, and a gift from Dr. Michael Green, University of Massachusetts Medical Center, Worcester, MA), anti-numatrin/B23 polyclonal antibody was generated as described previously (55). Glutathione cross-linked to agarose beads, creatine phosphokinase from rabbit muscle, inorganic phosphate from yeast, concanavalin A, phorbol 12-myristate 13-acetate, N-ethylamide, N-tosyl-L-phenylamine chloromethyl (TPCK), N-tosyllysine chloromethyl ketone (TLCK), and N-acetyl-Leu-Leu-norleucinal (LLnL) all were purchased from Sigma.

Yeast Two-hybrid Screen-- Full-length ATF2 cDNA (a generous gift from Dr. Michael Green), was subcloned in frame with the GAL4 DNA binding domain in the yeast expression vector pGBT9 (CLONTECH) at the BamHI site. The plasmid was confirmed to carry the ATF2 cDNA in the correct orientation and was transformed using a standard LiCl transformation procedure into the HF7C strain of yeast. The presence of ATF2-pGBT9 in the yeast was stably maintained by selection for the pGBT9 selection marker TRP1, which allows yeast growth in the absence of tryptophan. Expression of ATF2-pGBT9 only in the yeast cells transfected with ATF2-pGBT9 was confirmed by Western analysis using antibody to ATF2. HF7C yeast cells transformed only with pGBT9-ATF2 were then tested for activation of the reporter genes HIS3 and LacZ. It was seen that pGBT9-ATF2 alone or cotransformed with the activation domain (pGAD10) did not activate either the HIS3 or LacZ reporter genes, indicating that the pGBT9-ATF2 construct alone failed to activate at the GAL4 binding sites of the reporter genes. A PHA-stimulated human peripheral leukocyte Matchmaker^TM cDNA library (CLONTECH) was transformed into HF7C yeast already containing the pGBT9-ATF2 construct. Transformants were grown in the absence of tryptophan, leucine, and histidine (selective minimal media) at 30 °C for 8 days. The library was screened twice by this method with a total of 3 × 10⁶ clones analyzed. Colonies that grew in this selective minimal media condition (eight colonies), were scored for -galactosidase activity by the filter assay method. Colonies were lifted directly from plates onto Whatman no. 50 filters, lysed by freeze-fracture in liquid nitrogen, and allowed to thaw at room temperature for 1 min. Filters were then incubated, colony side up, on Whatman no. 3MM filters that had been presoaked in -galactosidase assay buffer. Filters were incubated at 30 °C for up to 6 h. His⁺/-galactosidase⁺ colonies were directly recovered from the plate and grown in selective media lacking leucine. DNA inserts from the positive yeast clones were electrotransformed into HB101 leucine auxotrophic bacterial cells. The HB101 transformants were grown on M9 (Leu, Amp) minimal medium to select for plasmids that contained the pGAD10 vector (Leu, Amp). The cDNA inserts were sequenced using automated sequencing analysis (ABI sequence analyzer, Kimmel Cancer Institute, Jefferson University).

Cloning of Full-length hUBC9 Gene Transcripts from gt10 Human Peripheral Blood Leukocyte cDNA Library-- A 670-bp probe was prepared from the coding region of the hUBC9 cDNA by EcoRI/SpeI digestion. This ³²P-labeled probe was used in Southern blot, Northern blot, and screening of gt10 human peripheral blood leukocyte cDNA library. cDNA inserts were excised from purified phage DNA by EcoRI digestion. From 1 × 10⁶ plaque-forming units that were screened with the 670-bp probe, two phages were obtained that were carried through primary, secondary, and tertiary screenings. These phages were grown in liquid culture, and a DNA extraction was performed. The cDNA inserts were released by EcoRI digestion of purified phage DNA and subjected to Southern blot analysis utilizing the 670-bp coding region probe. Both phage carried an identical insert of 1.3 kb that hybridized strongly to the probe. The inserts were sequenced using automated sequencing analysis (ABI sequence analyzer, Kimmel Cancer Institute, Jefferson University).

RNA Isolation and Northern Blot Analysis-- Total RNA was isolated using the guanidinium isothiocyanate method as described previously (6). 10 µg of total RNA was analyzed on glyoxal-agarose gel and transferred onto Nytran filters. Human multiple tissue Northern blots (CLONTECH) contained 2 µg of poly(A)⁺ RNA per lane of different human tissues run on a denaturing formaldehde 1.2% agarose gel, transferred to a charged modified nylon membrane, and fixed by UV irradiation. A 670-bp probe was prepared from the coding region of the hUBC9 cDNA by EcoRI/SpeI digestion and labeled by random priming using [³²P]dCTP. Hybridization was performed as described previously (6).

Southern Blot Analysis-- Southern blot (Zoo Blot, CLONTECH) contained 4 µg of genomic DNA per lane from eukaryotic different species. DNA was digested with EcoRI, run on a 7% agarose gel, transferred to a charged modified nylon membrane, and fixed by UV irradiation. A 670-bp probe was prepared from the coding region of the hUBC9 cDNA by EcoRI/SpeI digestion and labeled by random priming using [³²P]dCTP.

Generation of a Polyclonal Antibody to hUBC9-- The hUBC9 cDNA was subcloned into the pGEX2T vector in frame with the glutathione S-transferase (GST) moiety. The pGEX2T-hUBC9 construct was transformed into BL21 pLysS Escherichia coli and induced to express a GST-hUBC9 fusion protein by addition of isopropyl-1-thio--Dgalactopyranoside. The fusion protein was isolated and purified by affinity chromatography using glutathione-agarose beads. The fusion protein was then eluted from the beads and was sent for production of a polyclonal antibody in rabbit (horseradish peroxidase, Antisera Services, Denver, PA). An enzyme-linked immunosorbent assay, which was performed using purified hUBC9 (the GST moiety was cleaved using thrombin cleavage assay), demonstrated a titer of 1:204,800. Western blot analysis detected specifically an ~18-20-kDa protein.

Far Western Overlay Binding Assay-- This assay was performed using the protocol described previously (34). pGEX3X-ATF2 construct was transformed into BL21 E. coli and induced to express GST-ATF2. ATF2 was cleaved from the GST moiety by incubation with factor Xa (ICN) at 40:1 w/w ratio for 16 h at 4 °C in buffer containing 100 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1 mM CaCl₂. The GST moiety was pelleted using glutathione-agarose beads, and the supernatant was collected as purified ATF2. Purified ATF2 and purified actin (Sigma) were analyzed on SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated in blocking solution (5% bovine serum albumin in UB buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl and 0.1% NaN₃) overnight at 4 °C. The blocked membrane was incubated in overlay buffer, containing: 0.5% bovine serum albumin, 0.25% gelatin, 1% Nonidet-P40, 10 mM NaCl, 1 mM EGTA, 0.1% -mercaptoethanol, 20 mM HEPES, pH 7.5. GST-hUBC9 was radiolabeled with [-³²P]ATP in the presence of heart muscle kinase and exposed to thrombin cleavage as described (34). The purified radiolabeled hUBC9 (~20,000 cpm) was added to the overlay buffer and incubated for 4 h at room temperature. The membrane was then washed extensively in UB buffer supplemented with 0.5% sarcosyl, dried, and autoradiographed.

Purification of Nuclei and Western Blot Analysis of hUBC9-- Subcellular fractionation of nuclei and cytosolic fraction were performed as described previously (6, 35, 56). Jurkat T cells were suspended in RPMI 1640 at 2 × 10⁶ cells/ml and incubated in the presence or absence of concanavalin A (5 µg/ml) and PMA (10 ng/ml). At the end of the incubation period, the cells were washed with phosphate-buffered saline and resuspended in 100 µl/10 × 10⁶ cells of RSB buffer (0.01 M Tris-HCl, 0.01 M NaCl, 1.5 mM MgCl, pH 7.2) supplemented with 0.1% Nonidet P-40 and protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, for 2 min at 4 °C. The nuclei were pelleted by centrifugation at 4 °C, and the supernatants were collected as the soluble fraction and stored at 80 °C until further analysis. Nuclear proteins were extracted in urea lysis buffer containing 4.5 M urea, 1% Nonidet P-40, and 2.5% -mercaptoethanol. Equal amounts of nuclear or soluble proteins (30 µg) were mixed with Laemmli gel sample buffer, boiled for 5 min, analyzed by SDS-PAGE, and electrotransferred onto nitrocellulose membrane. The membrane was blocked by incubation in Tris-buffered saline containing 10 mM Tris, pH 7.2, 150 mM NaCl, and 5% nonfat dry milk for 1 h and then was blotted with anti-hUBC9 antiserum (1:1000 dilution) at room temperature for 2 h. Detection of the immunoreactive band was done by ECL system. Another cellular fractionation method was performed to verify no cross-contamination between the soluble and the nuclear fractions. Cells (~95 × 10⁶) were washed and then resuspended in 3 ml of homogenization buffer containing: 20 mM Tris, pH 7.4, 3 mM CaCl₂, 0.25 M sucrose and 0.2 mM phenylmethylsulfonyl fluoride. The cells were homogenized using Dounce homogenizer (20-30 strokes) at 4 °C. The presence of intact nuclei was verified using the microscope. The nuclei were pelleted by centrifugation at 2200 rpm at 4 °C for 5 min. The soluble fraction was further purified by centrifugation of the supernatant at 100,000 × g for 60 min at 4 °C. The supernatant was collected, and the proteins were acetone-precipitated.

Preparation of Cell Extract for in Vitro Ubiquitination-- Jurkat T cells at logarithmic growth were washed with phosphate-buffered saline and lysed in buffer containing 50 mM Tris, pH 7.5, 0.25 M sucrose, 0.1% Triton X-100, and 1 mM dithiothreitol. Cells were homogenized at 4 °C, the unbroken cells were pelleted rapidly in a microcentrifuge, and the supernatant was frozen at 20 °C until further use in ubiquitin conjugation assay.

Ubiquitin Conjugation Assay-- Purified GST-ATF2 was exposed to a standard ubiquitin conjugation assay as described previously (36, 37). The reaction mixture (35 µl) contained: 5-15 µg of GST-ATF2, 5-10 µg of ubiquitin, 50 mM Tris, pH 7.6, 5 mM MgCl, 2 mM ATP, 0.6 units/ml creatine phosphokinase from rabbit muscle (Sigma), 50 µM dithiothreitol, 0.6 units/ml inorganic phosphate from yeast (Sigma), 5 mM creatine phosphate, 10-50 µg of Jurkat T cell protein extract (as indicated in the specific experiments), purified hUBC9 (~150 ng), and 1 µM lactacystin (purchased from Dr. E. Corey, Harvard University, Cambridge MA). The reaction mixture was incubated at 37 °C for various periods of time as indicated and stopped by adding 20 µl of glutathione-agarose beads and incubation at 4 °C. After 7 min of incubation at 4 °C, the beads were pelleted by centrifugation at 4 °C, and washed twice more with Tris-buffered saline. The pelleted beads were dissolved in 30 µl of 2× SDS-sample buffer, boiled, and analyzed on 7.5% acrylamide gels. The proteins were electrotransferred onto nitrocellulose membrane, and the ubiquitin-conjugated ATF2 forms were detected by Western blot analysis with anti-ATF2 antibody or with affinity-purified antibody, which detect specifically ubiquitin conjugate. Western blot analysis of anti-Ub was done at 1:4000 dilution, and detection was performed with ECL.

Immunoprecipitation and Western Blot Analysis of ATF2-- Immunoprecipitation and Western blot were done as described previously (6, 55). Jurkat T cells were suspended at 1 × 10⁶ cells/ml and stimulated under different conditions as indicated. At the end of the incubation, the cells were washed with phosphate-buffered saline and lysed with RIPA buffer (0.15 M NaCl, 50 mM Tris, pH 8.0, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 5 mM EDTA, 1 mM dithiothreitol, leupeptine, phenylmethylsulfonyl fluoride, 0.25 mM TPCK, 0.25 mM TLCK, 5 mM N-ethylamide, 50 µM N-acetyl-Leu-Leu-norleucinal. Equal amounts of proteins (1-2 mg of proteins at 300 µl) were incubated with rotation at 4 °C with 1.5 µg of sheep anti-ATF2 IgG or nonimmune sheep IgG for 2-3 h. The immune complex was precipitated by incubation with 40 µl of protein G-agarose (Sigma) for 1 h. The agarose beads were pelleted by centrifugation and washed three times with RIPA. The beads were suspended in 2× SDS sample buffer, boiled, and analyzed on 10-12% acrylamide gel. Proteins were transferred onto nitrocellulose membrane and blotted with anti-ATF2 as described previously. In brief, after blocking with 5% nonfat dry milk, the membrane was incubated with anti-ATF2 (1:1000 dilution) and the antibody was detected using ECL detection system as described previously (4). In certain experiments, as indicated, the Western blot was performed after stripping the nitrocellulose membrane from a previously bound antibody. The nitrocellulose membrane was stripped by incubation in medium containing 2% SDS, 0.6% -mercaptoethanol, 62.5 mM Tris, pH 6.7, at 50 °C for 30 min with shaking. After extensive washing, the membrane was reblotted with anti-ATF2.

	RESULTS

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Cloning and Isolation of an ATF2-binding Protein in the Yeast Two-hybrid System Identifies a Novel Human Member of the Ubiquitin-conjugating Enzyme Family-- The two-hybrid interaction system in yeast was employed in an effort to isolate proteins that interact with the transcription factor ATF2. To this end, full-length ATF2 cDNA was subcloned in frame with the GAL4 DNA binding domain in the yeast expression vector pGBT9 at the BamHI site. The ATF2-pGBT9 plasmid was transformed into the HF7C strain of yeast containing stable constructs of the reporter genes LacZ and HIS3. The presence of ATF2-pGBT9 in the yeast was stably maintained by selection for the pGBT9 selection marker TRP1, which allows yeast growth in the absence of tryptophan. Expression of ATF2-pGBT9 only in the yeast cells transfected with ATF2-pGBT9 was confirmed by Western blot analysis using specific antibody to ATF2. HF7C yeast cells transformed with pGBT9-ATF2 were then tested for false activation of the yeast two-hybrid reporter genes (HIS3 and LacZ). pGBT9-ATF2 alone or cotransformed with the activation domain (pGAD10) alone did not activate either the HIS3 or LacZ reporter genes. This indicated that the ATF2 bait was not falsely activating at the GAL4 binding sites of these reporter genes. Thus, further experiments were performed to screen a human cDNA expression library of PHA-activated lymphocytes for proteins that interact with ATF2.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Predicted amino acid sequence of cDNA clone encoding a protein interactive with ATF2 in yeast two-hybrid system. Sequence shows identity to the recently reported hUBC9 and homology to several members of the ubiquitin-conjugating enzyme family. Alignment was done by using Bestfit program in the GCG package. Identical residues are printed in bold. Boxed area indicates the conserved active cysteine catalytic site, which is a conserved motif in members of the UBC family.

View larger version (57K): [in this window] [in a new window]   Fig. 2.   Expression of hUBC9 in stimulated T cells and in various human tissues. A, human peripheral blood T lymphocytes from a young adult donor were purified and stimulated with concanavalin A (2 µg/ml) for 24 or 48 h. At the end of the incubation, RNA was purified, analyzed on glyoxal-agarose gel (10 µg/lane), and transferred onto Nytran filters. B and C are human multiple tissue Northern blots (CLONTECH) containing 2 µg of poly(A)+ RNA/lane of different human tissues. The membranes were hybridized with a 32P-labeled 670-bp probe prepared from the coding region of the hUBC9 cDNA, or control probes: for Notch2 gene (kindly provided by Dr. C. Sell) or glyceraldehyde-3-phosphate dehydrogenase to verify equal amounts of RNA in the different lanes. Arrows indicate the 1.3-kb transcript of hUBC9 and a 3.0-kb transcript, which hybridized with the [32P]hUBC9 probe.

View larger version (67K): [in this window] [in a new window]   Fig. 3.   Southern blot analysis of hUBC9 gene in various species. Southern blot (Zoo Blot, CLONTECH) containing 4 µg of genomic DNA/lane from eukaryotic different species. The blot was hybridized with a [32P]hUBC9 670-bp probe prepared from the coding region of the hUBC9 cDNA and exposed to autoradiography.

Quantitative Analysis of hUBC9 Binding to ATF2 in the Yeast Two-hybrid Assay: Comparison with CREB and ATF1-- Further studies in the yeast two-hybrid system were pursued to quantitatively compare the level of interaction of hUBC9 with ATF2 to its interaction with other proteins (lamin, CREB, and ATF1) and to null vectors expressing the GAL4 DNA binding domain or the activation domain alone. CREB and ATF1 are members of the ATF/CREB family of transcription factors, which bind to CRE sequences in the promoters of genes but, in contrast to ATF2, may be regulated by the cAMP-dependent pathway (38). CREB and ATF1 full-length cDNA (kindly provided by Dr. C.-Z. Giam, Case Western Reserve University, Cleveland, OH) were cloned into the yeast expression vector pGBT9 within the open reading frame of the GAL4 (amino acids 1-147) DNA binding domain. Yeast cells of strain SFY526 containing the LacZ reporter gene were cotransformed with the hUBC9-pGAD10 plasmid and pGBT9-CREB, pGBT9-ATF1, pGBT9-lamin, or pGBT9-ATF2 as well as control plasmids. Transformants were grown on Trp/Leu yeast dropout plates at 30 °C for 4 days. Individual colonies were taken from each plate and grown overnight in liquid dropout medium at 30 °C. Equal amounts of cells were collected, and the activation of the reporter gene was measured by the quantitative liquid -galactosidase assay (Fig. 4A) as well as by -galactosidase filter assay (Fig. 4B).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Comparison of hUBC9 binding to ATF2, CREB, ATF1 and lamin. Yeast cells SFY526 containing the LacZ reporter gene were cotransformed with the hUBC9-pGAD10 plasmid and pGBT9-CREB, pGBT9-ATF1, pGBT9-lamin, or pGBT9-ATF2, as well as null plasmids. Transformants were grown on Trp/Leu yeast dropout plates at 30 °C for 4 days and then assayed for -galactosidase activity in filter assay (B). Individual colonies were taken from each plate and grown overnight in liquid dropout medium at 30 °C, equal amounts of cells were collected and further assayed for -galactosidase activity in liquid assay (A). Results represent the mean of (OD420/OD600) × 1000 of quadruplicate samples ± standard deviation. The experiment was performed two times with similar results.

Demonstration of hUBC9 Binding to ATF2 in the Filter Overlay Binding Assay-- The filter overlay binding assay is an assay for protein-protein interaction. This assay was previously shown to correlate with protein-protein interactions detected in the two-hybrid system (34). Thus, we have utilized this assay to examine the interaction of hUBC9 with ATF2. To this end, hUBC9 cDNA was subcloned into the pGEX2T vector in frame with the GST moiety. The pGEX2T-hUBC9 construct was transformed into BL21 pLysS E. coli and induced to express a GST-hUBC9 fusion protein. Purified GST-hUBC9 fusion protein was radiolabeled with [-³²P]ATP in the presence of heart muscle kinase (Fig. 5A). The GST moiety was cleaved from the hUBC9 protein by utilizing a thrombin cleavage site engineered between GST and hUBC9 (Fig. 5B). Thus, in this way, we have determined that hUBC9 has an apparent molecular mass of ~19 kDa as estimated by SDS-PAGE, which is consistent with the predicted amino acid sequence (Fig. 5B).

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Binding of hUBC9 to ATF2 in Far Western blot analysis. A, scheme of the pGEX-2T 128/129 vector fused to hUBC9 cDNA. cDNA of hUBC9 was subcloned into pGEX-2T 128/129 vector containing a heart muscle kinase site and a thrombin cleavage site in frame with the GST moiety. B, analysis of 32P-labeled thrombin-cleaved hUBC9 by SDS-PAGE. The pGEX2T-hUBC9 construct was transformed into BL21 pLysS E. coli and induced to express a GST-hUBC9 fusion protein. The fusion protein was purified using glutathione-agarose beads and was radiolabeled with [-32P]ATP in the presence of heart muscle kinase. The 32P-labeled GST-hUBC9 was further exposed to thrombin cleavage and analyzed by SDS-PAGE. C, [32P]hUBC9 binds specifically to ATF2 in Far Western blot analysis. Purified ATF2 and purified actin at various amounts were analyzed on two parallel SDS-PAGE gels. a, proteins were transferred onto nitrocellulose membrane and incubated with purified [32P]hUBC9 using the protocol of Far Western as described under "Materials and Methods." The membrane was washed extensively, dried, and autoradiographed. b, the gel was stained with Coomassie Blue staining.

Nuclear Localization of hUBC9-- We generated an antibody to GST-hUBC9 that detected purified hUBC9 by enzyme-linked immunosorbent assay at a titer of 1:205,000. This antibody detected a ~19-kDa protein in Western blot assays of protein extracts derived from murine lymph node, spleen, and thymus tissues and from Balb C 3T3 fibroblast cells (data not shown). To determine whether hUBC9 is found in the nuclei or translocated to the nuclei upon T cell activation, we purified nuclei from Jurkat T cells that were stimulated for 30 min with ConA+PMA. Equal amounts of the nuclear proteins and soluble proteins were analyzed on SDS-PAGE, transferred onto nitrocellulose, and blotted with anti-hUBC9 antibody. Fig. 6A demonstrates that hUBC9 has a molecular mass of ~19 kDa and is more abundant in the nuclei as compared with the soluble fraction in both control and stimulated T cells. Stimulation of Jurkat T cells with ConA+PMA for 30 min was not associated with any change in the subcellular localization of hUBC9. A further subcellular fractionation experiment was performed to determine the possibility of cross-contamination of the nuclear and soluble fractions. Fig. 6B demonstrates equal protein staining of the nuclear and soluble fractions of Jurkat T cells. Using Western blot analysis, we have demonstrated that actin is significantly more abundant in the soluble fraction. Conversely, numatrin (also called B23 and nucleophosmin), which we have shown previously to be exclusively localized in the nuclear fraction (35), was completely absent from the soluble fraction (even in higher exposure numatrin could not be detected in the soluble fraction (data not shown). Reprobing the blot with anti-hUBC9 demonstrates the presence of this protein in both fractions (whereas numatrin could only be detected in the nuclei fraction). Densitometry analysis showed that the amount of hUBC9 in the nuclei is 3.9-fold higher as compared with the amount of hUBC9 in the soluble fraction. This verifies that hUBC9 is predominantly a nuclear protein. The evidence of the presence of hUBC9 in the nuclei is important because it corroborates with a potential physiological significance to the interaction of hUBC9 with ATF2 or nuclear proteins.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Nuclear localization of hUBC9. A, Jurkat T cells were suspended to 2 × 106 cells/ml in RPMI 1640 and stimulated with ConA (5 µg/ml) and PMA (10 ng/ml) for 30 min. At the end of the incubation, the nuclear and cytosol fraction were purified using RSB buffer and equal amounts of proteins (30 µg) were analyzed on SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was blotted with anti-hUBC9 antibody (1:1000) dilution, and the immunoreactive protein was detected using the ECL system. B, Jurkat T cells were fractionated into soluble and nuclei fractions using sucrose containing homogenization buffer as described under "Materials and Methods," analyzed on SDS-PAGE, and transferred onto nitrocellulose membrane. The membrane was stained with Ponceau S (a) and further exposed to Western blot analysis with anti-actin (b), anti-numatrin (c), or anti-numatrin +anti-hUBC9 (d).

Evidence That ATF2 Is Ubiquitinated in Vivo and in Vitro-- Further experiments were undertaken to examine whether ATF2 is ubiquitinated in vivo. To this end, Jurkat T cells were incubated for 17 h with various doses of a compound frequently used as an inhibitor of the proteasome LLnL (39-41). ATF2 was then immunoprecipitated from protein extracts, analyzed by SDS-PAGE, and blotted with affinity-purified antibody specific for conjugated ubiquitin. Fig. 7A demonstrates a dose-dependent increase in ubiquitination of ATF2 in Jurkat T cells treated with LLnL. To enable detection of the nonubiquitinated ATF2 the membrane was stripped and reblotted with anti-ATF2. This analysis showed that the increase in the amounts of ubiquitinated forms of ATF2 was associated with a parallel dose-dependent decrease in the amounts of the major nonubiquitinated form of ATF2 (indicated as a in Fig. 7A), and a concomitant dose-dependent accumulation of degradation products of ATF2 (indicated as b and c in Fig. 7A). None of the various ATF2 forms were detected when protein extracts were precipitated with nonimmune IgG (see in Fig. 8C). The evidence of LLnL-induced ubiquitination of ATF2 in vivo and the concomitant accumulation of intermediate degradation products indicates that ATF2 may be a target of proteolytic processes involving the proteasome pathway.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Ubiquitination of ATF2 in vivo and in vitro. A, ubiquitination of ATF2 in vivo. Jurkat T cells were incubated for 17 h with various amounts of LLnL: lane 1, 0; lane 2, 25 µM; lane 3, 50 µM; lane 4, 100 µM. Cells were lysed in RIPA supplemented with 5 mM EDTA and 50 µM LLnL as described under "Materials and Methods" and immunoprecipitated with anti-ATF2. The immunoprecipitates were analyzed on SDS-PAGE, and the nitrocellulose membrane was blotted with antibody to ubiquitin conjugates. To determine the presence and position of nonubiquitinated ATF2, the nitrocellulose membrane was further stripped from the anti-Ub antibody as described under "Materials and Methods" and then reblotted with anti-ATF2. Arrows indicate the positions of the nonubiquitinated forms of ATF2: the major form (a) and the degradation forms (b and c). WB, Western blot. B, ubiquitination of ATF2 in vitro. Purified GST-ATF2 fusion protein (~15 µg) was exposed to standard ubiquitin conjugation assay using Jurkat T cell protein extracts (25 µg) and purified hUBC9 (150 ng) as a source of enzymes as described under "Materials and Methods." After incubation at 37 °C for 40 min, the GST-ATF2 was pelleted using glutathione beads, analyzed on SDS-PAGE, transferred onto nitrocellulose membrane, and blotted with anti-Ub antibody (left panel). To determine the presence and position of the nonubiquitinated ATF2, the nitrocellulose membrane was further reblotted with anti-ATF2 without stripping the Ub antibody (right panel). Arrows indicate the position of the nonubiquitinated forms of ATF2. C, ubiquitination of ATF2 in vitro is facilitated by hUBC9. Ubiquitin conjugation assay was performed as described under "Materials and Methods" using 5 µg of purified GST-ATF2 in the presence or absence of purified hUBC9 (150 ng) supplemented with 10 µg of Jurkat T cell extracts as a source of enzymes. The reaction was carried on for various periods of time from 0 to 180 min. GST-ATF2 was pelleted with the glutathione beads, analyzed on SDS-PAGE, and blotted with anti-ATF2 antibody. z indicates a protein that reacted nonspecifically in GST-ATF2 preparation with anti-ATF2 antibody.

View larger version (75K): [in this window] [in a new window]   Fig. 8.   ATF2 Degradation and phosphorylation are co-regulated upon T cell activation. A, Jurkat T cells were stimulated with ConA (5 µg/ml) and PMA (10 ng/ml) for various periods of time. At the end of the incubation, the cells were lysed with RIPA and equal amounts of proteins were used in immunoprecipitation (IP) with anti-ATF2 antibody or with nonimmune IgG. The immunoprecipitates were analyzed on SDS-PAGE, transferred onto nitrocellulose membrane and blotted with anti-ATF2 antibody. Arrows indicate the position of the major ATF2 form (a), minor degradation forms (b and c), and the phosphorylated form (p-a). B, Jurkat T cells were stimulated as in A in the presence of LLnL (25 µM). At the end of the incubation, the cells were lysed with RIPA buffer and equal amounts of proteins were used in immunoprecipitation with anti-ATF2 antibody. The immunoprecipitates were analyzed on SDS-PAGE, transferred onto nitrocellulose membrane, and blotted with anti-ATF2 antibody. Arrows indicate the major form of ATF2 (a), the minor degradative forms (b and c), and the phosphorylated form (p-a). C, Jurkat T cells were incubated with LLnL (25 µM) in the presence or absence of ConA+PMA for various periods of time. At the end of the incubation, the cells were lysed with RIPA and equal amounts of proteins were used in immunoprecipitation with anti-ATF2 antibody or with nonimmune IgG. The immunoprecipitates were analyzed on SDS-PAGE, transferred onto nitrocellulose membrane, and blotted with anti-ATF2 antibody. Arrows indicate the position of the major ATF2 form (a), degradation forms (b and c), and the phosphorylated form (p-a).

Evidence That Degradation and Phosphorylation of ATF2 Are Co-regulated in T Cells-- The finding that ATF2 interacts with a ubiquitin-conjugating enzyme and is ubiquitinated in vivo implicates the ubiquitin-proteasome pathway in regulation of ATF2. To explore this possibility, we investigated whether ATF2 is exposed to proteolytic processes during T cell activation. In Fig. 8 (A-C), cells were stimulated for various periods of time with ConA+PMA and the proteins were immunoprecipitated with anti-ATF2 or with nonimmune IgG. The immunoprecipitates were analyzed on gels and further blotted with anti-ATF2 in Western blot analysis. It was seen that there are no detectable changes in the abundance of the major form of ATF2 (indicated as a in Fig. 8A) in Jurkat T cells stimulated with ConA+PMA for various periods of time. However, minor lower molecular weight forms of ATF2 were detected specifically by ATF2 antibody in ATF2-immunoprecipitates and not in nonimmune IgG immunoprecipitates (Fig. 8A). These bands were found previously to be accumulated by LLnL (see Fig. 8A) and, thus, represent intermediate degradation products of ATF2. We now report that ATF2 degradation products are constitutively present in unstimulated T cells and reproducibly demonstrated a rapid decrease in abundance 30 min after T cell activation (Fig. 8A).

View larger version (35K): [in this window] [in a new window]   Fig. 9.   LLnL delays the down-regulation of ATF2 phosphorylation in stimulated T cells. Jurkat T cells were incubated in the presence or absence of LLnL (100 µM) for 1 h and then further incubated with ConA+PMA for various periods of time. Equal amounts of proteins were analyzed on SDS-PAGE, transferred onto nitrocellulose, and blotted with anti-ATF2 antibody. Arrows indicate the position of the major ATF2 form (a), a minor degradation form (b), and the phosphorylated form (p-a).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The yeast two-hybrid interaction system was employed to elucidate the mechanisms that regulate ATF2 during lymphocyte activation. We report a novel ATF2-interacting protein that is a new member of the ubiquitin-conjugating enzyme family, hUBC9. We demonstrate nuclear localization of hUBC9 and its interaction with two other CRE-binding transcription factors: CREB and ATF1. Evidence is presented to demonstrate that ATF2 is exposed to degradation processes in T cell and that it is ubiquitinated in vivo and in vitro. Taken collectively, this evidence implicates a role for hUBC9 and the proteasome pathway in regulation of ATF2 in T cells.

The ubiquitin-dependent degradation system is a major pathway in the selective degradation of proteins in eukaryotes. Evidence is accumulating to show that ubiquitination is involved in regulation of gene expression, DNA repair, cellular stress response, cell cycle progression, signal transduction, and programmed cell death (for review, see Refs. 31-33 and 43). In the ubiquitination reaction, ubiquitin, a highly conserved protein of 76 amino acids, is first activated by an activation enzyme, E1. It is then transferred to a cysteine residue of a ubiquitin-conjugating enzyme (E2 class). The E2 enzyme, either alone or together with a ubiquitin-protein ligase, E3, catalyzes transfer of ubiquitin to a lysine residue of a target protein. The ubiquitinated protein is recognized and degraded by a multisubunit 26 S proteasome complex. Ubiquitination of some proteins, such as calmodulin, histones, and certain membrane receptors, may serve a regulatory function without targeting them to cytosolic degradation (44).

The ubiquitin-conjugating enzymes (E2) are a family of proteins characterized by a highly conserved catalytic site containing an invariant active cysteine site (31-33). Previous studies in yeast Saccharomyces cerevisiae have identified at least 10 different UBCs (32) that are involved in various cellular processes such as DNA repair, cell cycle progression and in heat shock resistance, suggesting that E2 enzymes may be key players in establishing the diversity and the specificity of the ubiquitin-proteolytic system. Yeast UBC9 protein and its homolog, Hus5, have been shown to be essential for cell viability (32) and for normal mitosis (45). Repression of UBC9 synthesis in S. cerevisiae results in cell cycle arrest in G₂/M phase (46). In this case, UBC9 was implicated in the degradation of mitotic B-type cyclins, Clb5 and Clb2 (43). Although the sequence of the human homolog of UBC9 has been reported recently (28-30), the function of hUBC9 is not yet known.

In this work, the specificity and affinity of hUBC9 interaction with ATF2 in vivo was markedly demonstrated in a quantitative two-hybrid assay showing a high affinity of hUBC9 to ATF2 and lack of any interaction with lamin or with the GAL4 DNA binding domain. Intriguingly, hUBC9 demonstrated lower but significant interaction with CREB and ATF1, indicating the potential involvement of hUBC9 also in regulation of these transcription factors. In this respect, it is noteworthy that hUBC9 is demonstrated to localize primarily in the nucleus. Indeed, hUBC9 was originally identified via its interaction in yeast two-hybrid with other nuclear proteins: WI1 transcriptional repressor (29), Rad51 (28), and the human papillomavirus type 16 E1 (30). Although the nature and the function of this interaction remains to be investigated, it is possible that hUBC9 is an important ubiquitin-conjugating enzyme in the nucleus that plays a crucial role in regulation of nuclear proteins and particularly transcription factors.

Notably, the interaction of hUBC9 with ATF2 was ~10-fold higher as compared with CREB and ATF1, indicating that hUBC9 may have a more significant role in interaction with ATF2. The marked specificity of hUBC9 binding to ATF2 was further confirmed in vitro in the Far Western blot, showing that purified [³²P]hUBC9 binds to ATF2 protein, whereas it does not bind at all to actin.

Several lines of evidence indicates that the interaction of hUBC9 and ATF2 may have an important physiological significance. Most significant, by using an inhibitor of the proteasome, LLnL, we demonstrate that ATF2 is ubiquitinated in vivo. LLnL also induced a marked dose-dependent accumulation of intermediate degradation products of ATF2. We further demonstrate that the abundance of the ATF2 degradation products is tightly regulated during T cell activation. Specifically, we show that the ATF2 degradation forms are expressed constitutively in nonstimulated Jurkat T cells and that T cell activation is associated with a rapid and transient disappearance of these ATF2 degradation products. Because the presence of these degradation products represents a steady state equilibrium between the rate of their formation and the rate of their final processing, the evidence of their disappearance indicates that, upon T cell activation, there is an interference in this steady state equilibrium. Thus, upon T cell activation, there is either a marked increase in the final processing of these degradation forms or a marked decrease in the formation of these degradation products via degradation of the major ATF2 forms.

Intriguingly, the change in the abundance of the ATF2 proteolytic products was concomitant with the induction of phosphorylation of the major ATF2 form. Thus, it is tempting to speculate that induction of phosphorylation of ATF2 may have an effect on its rate of degradation. ATF2 was shown to be rapidly phosphorylated in response to several external stimuli such as growth factors and UV irradiation (23-26). This phosphorylation is mediated by JNK, a member of the MAP kinase family, in a process that was shown to involve physical binding of JNK to ATF2. Thus, it is possible that binding of ATF2 to hUBC9 may affect its ability to bind to JNK and vice versa. Indeed, a recent study demonstrated that phosphorylation of c-Jun by JNK or MAP kinase is accompanied by a reduction in c-Jun ubiquitination and consequently degradation (47). Thus, phosphorylation of c-Jun stabilizes the protein by inhibiting its ubiquitination (47). This evidence may be particularly pertinent in view of several lines of similarity between c-Jun and ATF2. Jun and ATF2 interact via heterodimer complexes in binding to either CRE or TPA-responsive element consensus sequences in the promoter of genes (see Refs. 38 and 48 and references therein). The activation of both c-Jun (Ref. 49; see also Ref. 26 and references therein) and ATF2 (23-26, 50) is mediated via phosphorylation, which occurs after activation by growth factors and UV radiation and is catalyzed by the MAP kinase family pathways. In addition, extensive evidence indicates that c-Jun is regulated by ubiquitination (47, 53, 54). Most recently, it has been reported that hUBC9 interacts specifically with the C-terminal half of c-Jun but not with c-Fos in yeast two-hybrid (57), indicating that hUBC9 may be the enzyme that regulates the ubiquitination of c-Jun. Thus, our studies indicate another line of similarity between ATF2 and c-Jun; both may be regulated by ubiquitination, which may involve hUBC9. The evidence that c-Jun phosphorylation stabilizes the protein by inhibiting its ubiquitination may corroborate with the observations of this work and suggests that a cross-talk between phosphorylation and degradation processes may be another similar characteristic of both c-Jun and ATF2. This possibility should be investigated in future studies.

Because there is no change in the abundance of the major ATF2 form during T cell activation, a question of importance is: what is the physiological function of the apparent proteolytic process of ATF2? Recent studies have brought to attention the crucial role of the ubiquitin-dependent degradation in regulation of signal transduction of discrete transcription factors (for review, see Ref. 51). Specifically, studies demonstrated the role of ubiquitination in regulation of the transcription inhibitor IB (Ref. 52; see Ref. 43, and references therin), the transcription factors p53 (Ref. 51, and references therein), STAT (42), and c-Jun (47, 53, 54). A special cross-talk between ubiquitination and phosphorylation has been demonstrated in previous studies related to IB, Jun, STAT, and TCR (42, 47, 52, 58). Ligand-dependent ubiquitination of T cell antigen receptor was shown to be dependent on its tyrosine phosphorylation by p56^lck (58). Furthermore, the tyrosine phosphatase CD45 is also necessary for antigen occupancy-stimulated TCR ubiquitination (58). Ubiquitination of the transcription factor STAT has been suggested to be responsible for negative regulation of the phosphorylation signal (42). In this respect, it is interesting that LLnL was found to inhibit the down-regulation of the phosphorylation of ATF2. This suggests that proteolytic processes may facilitate negative regulation of the phosphorylated ATF2 at a later time after T cell activation and, thus, provides a further evidence for a potential cross-talk between phosphorylation and degradation processes in regulation of ATF2. Finally, we further show that ATF2 can be ubiquitinated in vivo and also in vitro, and that its ubiquitination in vitro is facilitated by purified hUBC9. Taken collectively with the evidence in yeast two-hybrid and Far Western analysis, the data implicate a role for hUBC9 and the proteasome pathway in regulation of ATF2 in T cells.