Modification of CCAAT/Enhancer-binding Protein-β by the Small Ubiquitin-like Modifier (SUMO) Family Members, SUMO-2 and SUMO-3
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m305680200
ISSN1083-351X
Autores Tópico(s)NF-κB Signaling Pathways
ResumoCCAAT/enhancer-binding protein-β (C/EBPβ) activator isoforms, C/EBPβ-1 and C/EBPβ-2, differ by only 23 amino acids in the human; however, evidence is accumulating that these transcription factors are functionally distinct. Here we demonstrate that C/EBPβ-1, but not C/EBPβ-2, is conjugated to the small ubiquitin-like modifier (SUMO) family members, SUMO-2 and SUMO-3 despite the fact that the SUMO target consensus is present in both isoforms of this transcription factor. This conjugation is dependent on the integrity of the extreme N terminus of C/EBPβ-1 and requires lysine 173 in the human protein. Furthermore, mutation of this lysine relieves the repression of the cyclin D1 promoter by C/EBPβ-1 without altering the subnuclear localization of C/EBPβ-1. The sumoylation of C/EBPβ-1 is likely to be important in the functional differences observed between C/EBPβ-1 and C/EBPβ-2. CCAAT/enhancer-binding protein-β (C/EBPβ) activator isoforms, C/EBPβ-1 and C/EBPβ-2, differ by only 23 amino acids in the human; however, evidence is accumulating that these transcription factors are functionally distinct. Here we demonstrate that C/EBPβ-1, but not C/EBPβ-2, is conjugated to the small ubiquitin-like modifier (SUMO) family members, SUMO-2 and SUMO-3 despite the fact that the SUMO target consensus is present in both isoforms of this transcription factor. This conjugation is dependent on the integrity of the extreme N terminus of C/EBPβ-1 and requires lysine 173 in the human protein. Furthermore, mutation of this lysine relieves the repression of the cyclin D1 promoter by C/EBPβ-1 without altering the subnuclear localization of C/EBPβ-1. The sumoylation of C/EBPβ-1 is likely to be important in the functional differences observed between C/EBPβ-1 and C/EBPβ-2. The small ubiquitin-like modifier, or SUMO, 1The abbreviations used are: SUMO, small ubiquitin-like modifier; HA, hemagglutinin; C/EBP, CCAAT/enhancer-binding protein; LAP, liver-enriched activator protein; PBS, phosphate-buffered saline. proteins are a group of polypeptides that are conjugated to lysine residues in target proteins in much the same manner as ubiquitin (for a review, see Ref. 1Wilson V.G. Rangasamy D. Exp. Cell Res. 2001; 271: 57-65Crossref PubMed Scopus (86) Google Scholar). To date, three family members have been identified in higher eukaryotes: SUMO-1, SUMO-2, and SUMO-3. In humans and mice, the three isoforms appear to be ubiquitously expressed. SUMO-1 has been the most extensively characterized; homologues have been found in organisms ranging from yeast to humans. Glycine 97 in this 101-amino-acid protein is conjugated to a lysine in target proteins (2Mahajan R. Gerace L. Melchior F. J. Cell Biol. 1998; 140: 259-270Crossref PubMed Scopus (239) Google Scholar, 3Kamitani T. Nguyen H.P. Kito K. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 3117-3120Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) utilizing activating and conjugating enzymes similar to those in the ubiquitin conjugation pathway (4Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). The consensus sequence, (I/L)K XE, for sumoylation has been defined (see Ref. 5Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar for a review). Targets of SUMO-1 include the GTPase-activating protein RanGAP1 (10Lee G.W. Melchior F. Matunis M.J. Mahajan R. Tian Q. Anderson P. J. Biol. Chem. 1998; 273: 6503-6507Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), the promyelocytic leukemia gene product (PML) (6Sternsdorf T. Jensen K. Will H. J. Cell Biol. 1997; 139: 1621-1634Crossref PubMed Scopus (291) Google Scholar), IκBα (7Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar), the transcription factors AP-2 (8Eloranta J.J. Hurst H.C. J. Biol. Chem. 2002; 277: 30798-30804Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and several members of the C/EBP family (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Sumoylation of these proteins leads to localization of RanGAP1 at the nuclear pore (5Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar, 10Lee G.W. Melchior F. Matunis M.J. Mahajan R. Tian Q. Anderson P. J. Biol. Chem. 1998; 273: 6503-6507Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and localization of promyelocytic leukemia gene product into discrete bodies in the nucleus (5Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (656) Google Scholar, 6Sternsdorf T. Jensen K. Will H. J. Cell Biol. 1997; 139: 1621-1634Crossref PubMed Scopus (291) Google Scholar). Modification of IκBα by SUMO antagonizes ubiquitination and subsequent degradation of this inhibitor of the transcription factor NFκB (7Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). Recently, the repression domain I of C/EBPϵ has been demonstrated to be modified by SUMO-1 (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar); this modification is proposed to be important for the inhibitory function of this domain. These authors also show that conserved SUMO target sequences are present in C/EBPα, C/EBPβ, and C/EBPδ and that these isoforms can be conjugated to SUMO-1 (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). SUMO-2 and SUMO-3 are 66% homologous to SUMO-1; SUMO-2 and SUMO-3 are 97% identical to each other (11Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (419) Google Scholar). Despite the fact that the SUMO family members are very closely related, SUMO-2 and SUMO-3 have the ability to form poly(SUMO) chains owing to the fact that their amino acid sequence contains a SUMO target consensus (4Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). Because SUMO-1 does not contain a target lysine for sumoylation, it appears to be conjugated to proteins as a monomer (12Bayer P. Arndt A. Metzger S. Mahajan R. Melchior F. Jaenicke R. Becker J. J. Mol. Biol. 1998; 280: 275-286Crossref PubMed Scopus (327) Google Scholar). Antibodies currently available cannot distinguish between SUMO-2 and SUMO-3 (4Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar), but it is assumed that since SUMO-2 and -3 have such a high degree of sequence similarity, the two proteins have overlapping functions. Elucidation of their differences awaits further study. Little is known about the function of SUMO-2/-3 within cells. Saitoh and Hinchey (13Saitoh H. Hinchey J. J. Biol. Chem. 2000; 275: 6252-6258Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar) described that oxidative stress, heat shock, or UV irradiation caused a dramatic increase in the amount of SUMO-2/-3 incorporated in high molecular weight complexes within Cos-7 cells. At the current time, the identities of the proteins modified by SUMO-2/-3 in response to stress are unknown. C/EBPβ is a member of the basic leucine zipper family of transcription factors. The protein is transcribed from an intronless gene but gives rise to three protein isoforms due to alternative translation initiation at three in-frame ATG initiator codons (14Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (861) Google Scholar). In the human, the full-length C/EBPβ begins at the first in-frame methionine and consists of the entire 346 amino acids (this is 297 in the rat and mouse). The second isoform begins at the second in-frame methionine, which is 23 amino acids (or in rat/mouse, 21 amino acids) downstream from the first. The third isoform begins at the final in-frame methionine that is at position 198 in humans. The structure of C/EBPβ is such that the transactivation domain resides in the N-terminal region. The DNA binding and protein dimerization domains, the basic region and leucine zipper, reside in the C-terminal end. The first two isoforms consist of both the activation and DNA binding/dimerization domains and differ only by a 23-amino-acid N-terminal truncation. The third isoform, however, lacks the N-terminal activation domain while retaining the DNA binding/dimerization domain. This protein acts as a transcriptional repressor due to the fact that it can homodimerize or heterodimerize with the larger C/EBPβ isoforms and/or occupy the same C/EBPβ DNA elements within promoters of target genes. We have previously observed that C/EBPβ-1 and C/EBPβ-2 are differentially expressed between non-dividing, normal breast cells and dividing breast cells, either normal or neoplastic (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). Furthermore, introduction of C/EBPβ-2 into the normal, immortal mammary epithelial cell line, MCF10A, causes anchorage-independent growth, acquisition of invasive potential, and transition from an epithelial to mesenchymal state (16Bundy L. Sealy L. Oncogene. 2003; 22: 869-883Crossref PubMed Scopus (103) Google Scholar). This phenotype is not observed with the introduction of C/EBPβ-1. Kowenz-Leutz and Leutz (17Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) have demonstrated that C/EBPβ-1 (in their work, the rat protein termed LAP*) could recruit the chromatin remodeling complex Swi-Snf to the promoters of genes important in the differentiation of myeloid cells. The second isoform, C/EBPβ-2 or LAP, was unable to perform this function (17Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Our results, along with results from other laboratories, support the theory that these two activator isoforms, despite differing by only 21–23 amino acids, behave very differently within cells. Here, we demonstrate that C/EBPβ is modified by the SUMO family members SUMO-2 and SUMO-3. Furthermore, this modification is specific to the first isoform of C/EBPβ, or C/EBPβ-1, as the second activator isoform, or C/EBPβ-2, is not sumoylated. We show that lysine 173 in the human protein is the target of sumoylation by SUMO-2 and SUMO-3. Mutation of this lysine does not affect the punctate nuclear localization of C/EBPβ-1; however, this lysine is critical for the failure of C/EBPβ-1 to activate the cyclin D1 promoter. In addition, we demonstrate that the modification of C/EBPβ-1 by SUMO is dependent on the integrity of the N-terminal amino acids present in C/EBPβ-1, absent in C/EBPβ-2. Expression Vectors—Hemagglutinin (HA)-tagged SUMO-2 and HA-tagged SUMO-3 expression vectors were a kind gift of Dr. Ron Hay (University of St Andrews, St Andrews, UK). The expression vectors for T7-tagged C/EBPβ-1 and T7-tagged C/EBPβ-2 have been described previously as has the pGL3-cyclin D1 reporter construct and the empty CMV4 vector (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar, 16Bundy L. Sealy L. Oncogene. 2003; 22: 869-883Crossref PubMed Scopus (103) Google Scholar). The C/EBPβ-1 lysine 173 to alanine mutant was constructed as follows: an expression vector encoding a T7-tagged, 541-base pair C terminus mutant of C/EBPβ beginning at the first methionine (pRSETB LAP541, described previously in Ref. 18Sears R.C. Sealy L. Mol. Cell. Biol. 1994; 14: 4855-4871Crossref PubMed Google Scholar) was digested with EagI and HindII to release an 80-nucleotide fragment encompassing lysine 173. The vector was purified from the nucleotide fragment using gel electrophoresis. An oligonucleotide identical to the wild-type fragment except for an aag (Lys) to gcg (Ala) substitution was ligated into the pRSETB LAP541 vector. The sequence of the oligonucleotide, which was synthesized by Sigma-Genosys Corp., is as follows: 5′-ggc cgc gcg ggc gcc aag gcc gca ccg ccc gcc tgc ttc ccg ccg ccg cct ccc gcc gca ctc GCG gcc gag ccg ggc ttc ga-3′. Confirmation of the clone was obtained by sequencing. The full-length pRSETB LAP Lys173 → Ala mutant was constructed by digesting wild-type pRSETB LAP (described in Ref. 18Sears R.C. Sealy L. Mol. Cell. Biol. 1994; 14: 4855-4871Crossref PubMed Google Scholar) with BstBI to release the ∼900-base pair fragment absent in pRSETB LAP541. The fragment was isolated from the vector using gel electrophoresis and then ligated into the linearized, BstBI-digested pRSETB LAP541 Lys173 → Ala mutant. Orientation of the insert was determined using a diagnostic PstI digest (an 810-base pair fragment released if correct orientation, a 540-base pair fragment released if incorrect orientation). The LAP Lys173 → Ala mutant was placed into the pcDNA3.1hisB mammalian expression vector in the following manner. The pRSETB LAP Lys173 → Ala was first digested with HindIII to linearize; following heat inactivation of the enzyme, DNA polymerase I was used (at 4 °C) to fill in the 5′ overhang and create a blunt end. The construct was next digested with BamHI to liberate a ∼1.5-kb fragment. This fragment was purified via gel electrophoresis and ligated into the pcDNA3.1hisB expression vector that had been digested with BamHI and EcoRV. Confirmation of the correct clone was obtained by sequencing. The N-terminal mutant of C/EBPβ-1 was obtained as follows. The pRSETC NFIL-6 vector (which has been described in Ref. 18Sears R.C. Sealy L. Mol. Cell. Biol. 1994; 14: 4855-4871Crossref PubMed Google Scholar) was digested with BglII and MscI to release a 75-base pair fragment encompassing the N-terminal 23 amino acids of C/EBPβ-1. A replacement oligonucleotide was synthesized (Sigma-Genosys) that encoded for four amino acid mutations within the N terminus. The sequence of the oligonucleotide (with mutations in capitals) is: 5′-cc atg caa cgc ctg gtg gcc tgg AAA ggt gca GGT CGT ccc ctg ccg ccg ccg ccg cct gcc ttt aaa tcc GGA gaa gtg g-3′. After isolating the vector away from the wild-type fragment by gel electrophoresis, the mutant oligo was ligated into the vector. Confirmation of the clone, pRSETC-N-terminal mutant-NF-IL6, was obtained by sequencing. The N-terminal mutant C/EBPβ-1 was placed into the mammalian expression vector pcDNA3.1hisA as follows. The pRSETC-N-terminal mutant-NF-IL6 was digested with HinDIII, the restriction enzyme was heat-inactivated, and the overhang was blunt-ended with DNA polymerase I at 4 °C. The linearized vector was next digested with BamHI to release the fragment encoding the N-terminal mutant of C/EBPβ-1. This fragment was ligated into pcDNA3.1hisA digested with BamHI and EcoRV. Cells and Culturing Conditions—Cos-7 cells were a gift from Dr Steve Hann, Vanderbilt University. The cells were maintained in Dulbecco's modified Eagle's medium plus 10% calf serum (Colorado Serum Co., Denver, CO). Human mammary epithelial cells (HMECs) were purchased from Clonetics (Walkersville, MD) and maintained in mammary epithelial cell growth medium from the same company. NIH3T3 cells were purchased from the ATCC and maintained in Dulbecco's modified Eagle's medium plus 10% Colorado calf serum. All cells were kept in a humidified chamber at 37 °C with 5% CO2. Immunofluorescence—HMECs were transfected with 5 ng of expression vectors of C/EBPβ-1 or C/EBPβ-1 Lys to Ala mutant using Geneporter (GeneTherapy Systems, Inc.) as per the manufacturer's instructions. After 24 h, cells were plated onto polylysine-coated coverslips. Cells were allowed to sit down onto the coverslips and then fixed using 3.7% formaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and blocked with 5% bovine serum albumin in PBS. Coverslips were incubated with T7 tag antibody (Novagen, Madison, WI) at a 1:10,000 dilution in 2% bovine serum albumin, 0.1% Triton X-100 in PBS for 1 h at room temperature. Coverslips were washed extensively with 0.1% Triton X-100 in PBS and then incubated with goat anti-mouse secondary antibody conjugated to Alexa 594 (Molecular Probes, Eugene OR) at a 2 μg/ml dilution in 2% bovine serum albumin, 0.1% Triton X-100 for 1 h at room temperature in the dark. Coverslips were again washed extensively with 0.1% Triton X-100 in PBS and then rinsed with deionized water before staining with Hoechst dye at a 1 μg/ml in PBS concentration for 20 min at room temperature. Coverslips were washed with PBS and then mounted onto slides using Poly-Aquamount (Polysciences, Inc., Warrington, PA). Fluorescence was visualized using a Zeiss axiophot upright fluorescence microscope equipped with a low light CCD camera. Transfections and immunoprecipitations—NIH3T3 cells were transfected with either 2 μg of expression vector only (CMV4) or 2 μg of C/EBPβ-1, C/EBPβ-2, or C/EBPβ-1 Lys to Ala mutant expression vectors plus 2 μg of pGL3-cyclin D1 reporter vector using Novafector liposomes (VennNova, Pompano Beach, Fl). Luciferase assays were carried out as described previously (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). A portion of the lysates was combined with 2× SDS sample buffer, boiled, and subjected to 10% SDS-PAGE. Western analysis was performed as described previously (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar) using the T7 tag antibody at a 1:20,000 dilution. Cos-7 cells were transfected with 5 μg of indicated expression vectors using NovaFector liposomes as per the manufacturer's instructions. After 30–36 h, cells were harvested at 4 °C in PBS plus 0.1 mm sodium vanadate, spun at 840 × g, and resuspended, on ice in radioimmune precipitation buffer (20 mm Tris, pH 7.5, 50 mm NaCl, 0.5% Triton X-100, 0.5% sodium desoxycholate, 0.1% SDS, 1 mm EDTA) plus protease and phosphotase inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 10 mm sodium molybdate, 0.1 mm sodium vanadate, 10 mm β-glycerol-phosphate). Cell extracts were sonicated and added to T7 monoclonal antibody-conjugated agarose beads (Covance, Berkley, CA) that were washed previously with radioimmune precipitation buffer plus protease/phosphatase inhibitors. Following an overnight incubation at 4 °C, the beads were collected, extensively washed, and subjected to immunoblotting as described previously (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). Anti-HA polyclonal antibody from Clontech was used to probe the membranes at a 1:500 dilution. Anti-C/EBPβ antibody (Santa Cruz Biotechnology) was used at a 1:2000 dilution. Anti-T7 tag antibody (Novagen) was used at a 1:1000 dilution. Membranes were stripped using Re-Blot Plus from Chemicon International (Temecula, CA). HMECs were radiolabeled with [35S]methionine, and immunoprecipitations were performed as described previously (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar) with the exception being that guinea pig immune serum, raised to the full-length C/EBPβ, described in Ref. 18Sears R.C. Sealy L. Mol. Cell. Biol. 1994; 14: 4855-4871Crossref PubMed Google Scholar, was utilized. SUMO-2 and SUMO-3 Are Conjugated to C/EBPβ-1, but Not C/EBPβ-2—Given the fact that we observed a consensus SUMO-targeting sequence in C/EBPβ (this observation was confirmed while this work was in progress (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar)), we decided to investigate whether the two activator isoforms could be modified by SUMO-2/3. We utilized Cos-7 cells to perform co-transfection experiments with expression vectors for HA-tagged SUMO-2 or HA-SUMO-3 together with T7-tagged C/EBPβ-1 or C/EBPβ-2. Following the transfection, proteins were immunoprecipitated using the T7 monoclonal antibody conjugated to agarose beads. The immunoprecipitates were then subjected to immunoblotting with an HA tag antibody. As seen in Fig. 1A, HA-tagged SUMO-2 and HA-SUMO-3 were incorporated into C/EBPβ-1 (lanes 1 and 2) but not into C/EBPβ-2 (lanes 3 and 4). This disparity in incorporation occurs despite the fact that equivalent amounts of the C/EBPβ-1 and -2 proteins are expressed and immunoprecipitated in our system as shown when the blot in Fig. 1A was stripped and reprobed with anti-C/EBPβ antibody (Fig. 1B). The anti-C/EBPβ antibody also detects larger molecular weight bands when C/EBPβ-1 and SUMO-2 or -3 are expressed but not when C/EBPβ-2 and SUMO-2 or -3 are expressed (Fig. 1B), consistent with sumoylation of C/EBPβ-1 Interestingly, the apparent molecular mass of the sumoylated C/EBPβ-1 is ∼82 kDa (Fig. 1, A and B, lanes 1 and 2). This indicates that there are likely at least three of the ∼10–11-kDa SUMO proteins incorporated in C/EBPβ-1 (the apparent molecular mass of C/EBPβ-1 in our SDS-PAGE system is 55 kDa). Disruption of the N-terminal Extension of C/EBPβ-1 Reduces SUMO Incorporation—As stated earlier, the two activator isoforms of human C/EBPβ differ only by a 23-amino-acid truncation of the extreme N terminus of C/EBPβ-1. We next wanted to ascertain whether these amino acids were important for the striking difference we observed between the sumoylation of C/EBPβ-1 and C/EBPβ-2. To address this question, we made mutations in three amino acids, conserved between species, of the N-terminal extension of C/EBPβ-1 (Table I). We next performed immunoprecipitation experiments utilizing this mutant together with the wild-type construct. As seen in Fig. 2, mutation of these amino acids greatly reduced conjugation of SUMO-2/-3 to C/EBPβ-1. In Fig. 2, A and C, lanes 3 and 4 depict tagged C/EBPβ-1 and the tagged C/EBPβ-1 N-terminal mutant transfected into Cos-7 cells together with SUMO-2 (A) and SUMO-3 (C), immunoprecipitated with T7 antibody, and immunoblotted with an HA tag antibody. In this particular experiment, poly-sumoylated wild-type C/EBPβ-1 containing even more than three chains (Fig. 2, C, lane 3, higher molecular mass species of ∼116 kDa) is observed. However, mutation of the N-terminal amino acids markedly reduces the ability of both SUMO-2 and SUMO-3 to be incorporated into C/EBPβ-1 despite the fact that equivalent amounts of the C/EBPβ proteins are expressed and immunoprecipitated (as seen in the same blots (Fig. 2, B and D), stripped, and reprobed with T7 tag antibody). These data indicate that the integrity of the N terminus of C/EBPβ-1 is crucial for sumoylation.Table ISequence comparison of the N terminus of C/EBPβ-1SpeciesN-terminal sequenceHumanMQRLVAWDPACLPLPPPPPAFKSMMouseMHRLLAWDAACLPPPPAAFRPMRatMHRLLAWDAACLPPPPAAFRPMChickenMQRLVAWDAACLPIQPPAFKSMXenopusMHRLPQWDQAAACLPPPPGIRSMN-terminal mutant (human)MQRLVAWKPAGRPLPPPPPAFKSM Open table in a new tab Lysine 173 Is a Target of SUMO-2/-3—With the evidence that C/EBPβ-1 is being modified by SUMO-2/-3, we next wanted to ascertain whether the consensus lysine, at position 173 in the human protein, was the primary target. We developed a mutant form of C/EBPβ-1, in which the lysine 173 was changed into an alanine. Utilizing this construct in co-transfection experiments, we observed an ablation of incorporation of SUMO-2/-3 into the C/EBPβ-1 protein. As seen in Fig. 2, A and C, lanes 5, there is no incorporation of SUMO-3 and very little incorporation of SUMO-2 into the lysine 173 to alanine mutant despite the fact that the proteins are synthesized and immunoprecipitated (Fig. 2, B and D). This slight amount of SUMO-2 incorporated into the C/EBPβ-1 Lys to Ala mutant may represent sumoylation at a cryptic site within the protein. These results serve to indicate that lysine 173 is the critical residue being modified by SUMO-2/-3. Antibody Directed to C/EBPβ Immunoprecipitates a 97-kDa Protein from HMECs—Work from our laboratory has demonstrated previously that normal HMECs placed in culture from reduction mammaplasty express both activator isoforms of C/EBPβ (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). Antibody directed to the entire C/EBPβ protein immunoprecipitates proteins consistent with the molecular weight of C/EBPβ-1 (p55) and C/EBPβ-2 (p42) from [35S]methionine radiolabeled HMECs (Fig. 3). This antibody also immunoprecipitates a 97-kDa form of C/EBPβ from these cells. Because this isoform is larger than the sumoylated C/EBPβ-1 observed in Fig. 1 (in which both C/EBPβ-1 and the SUMO proteins carry epitope tags), it is likely that the C/EBPβ we observe in these cells is conjugated to more SUMO chains than the exogenously expressed C/EBPβ in Fig. 1 but could correspond to the higher molecular weight SUMO-3-conjugated species seen in Fig. 2C, lane 3 (which likely carries 1 T7 and 4 HA epitope tags). Lysine 173 Does Not Affect Nuclear Localization of C/EBPβ-1—SUMO proteins are known to affect cellular localization of target proteins. We have observed that C/EBPβ-1 has a specific, punctate nuclear staining pattern. 2E. M. Eaton and L. Sealy, unpublished observations. We hypothesized that the sumoylation of C/EBPβ-1 may be responsible for targeting the transcription factor to these areas. To address this question, we transfected HMECs with expression vectors for either wild-type C/EBPβ-1 or the C/EBPβ-1 lysine 173 to alanine mutant. Very small quantities (5 ng) of DNA were used to ensure that exogenous protein did not overwhelm the SUMO conjugation capacity of the cells. Transfected protein expression levels reflected those seen with endogenous C/EBPβ-1 protein (data not shown). As seen in Fig. 4, the staining pattern of the wild-type protein (C) mirrored that of the lysine 173 to alanine mutant (A). Hoechst staining demonstrates that the expression of C/EBPβ-1 is nuclear (Fig. 4, B and D). Thus, it appears that mutation of the sumoylation target does not affect subnuclear localization of C/EBPβ-1. Lysine 173 Is Critical for the Failure of C/EBPβ-1 to Activate the Cyclin D1 Promoter—Previously, we demonstrated that C/EBPβ-2, but not C/EBPβ-1, can activate the cyclin D1 promoter (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). Recently, Kim et al. (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) demonstrated that the lysine targeted by SUMO-1 (lysine 134 in the rat protein) was necessary for proper function of the repression domain I in a C/EBPβ-Gal4 fusion protein reporter assay. We wanted to investigate whether the conserved human lysine 173 was necessary for the failure of C/EBPβ-1 to activate this promoter. Using transient transfections in NIH-3T3 murine fibroblasts, we observed that mutation of this lysine caused C/EBPβ-1 to behave similarly to C/EBPβ-2 using the cyclin D1 promoter/luciferase reporter construct. Whereas C/EBPβ-2 activates this construct above basal levels (CMV4), C/EBPβ-1 does not (Fig. 5). Mutation of lysine 173 in C/EBPβ-1 results in activation of the cyclin D1 construct. All transactivator proteins were expressed at equivalent levels as shown in Fig. 5B. These results indicate that lysine 173 is critical for the function of the repression domain of C/EBPβ-1 using this promoter construct. C/EBPβ-1 and C/EBPβ-2 differ by only 21–23 amino acids, yet evidence continues to accumulate that they are functionally distinct. In 1999, Kowenz-Leutz and Leutz (17Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) demonstrated that the largest form of C/EBPβ, in their work termed LAP*, was capable of recruiting the Swi-Snf chromatin remodeling complex to the promoters of differentiation-specific genes in myeloid cells. The second isoform, LAP or C/EBPβ-2, was unable to recruit this complex to activate genes within the chromatin context. Furthermore, these authors demonstrated that the extreme N terminus of full-length C/EBPβ was necessary, but not sufficient, to recruit the Swi-Snf complex (17Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Our laboratory has found differences in the expression and function of C/EBPβ-1 and C/EBPβ-2 in mammary epithelial cells. We have demonstrated that C/EBPβ-2 is associated with dividing cells, either normal or cancerous, and that overexpression of this isoform causes transformation of normal mammary epithelial cells in culture (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). This transformation is not observed with introduction of C/EBPβ-1; moreover, we have demonstrated that this isoform is lost from breast cancer cell lines. The work presented here expands on the observation that C/EBPβ-1 and C/EBPβ-2 are functionally distinct by demonstrating that C/EBPβ-1, but not C/EBPβ-2, can be conjugated to the SUMO family members, SUMO-2 and SUMO-3. The intact N terminus of C/EBPβ-1 is needed, at least in part, for this conjugation, as evidenced by the reduction of SUMO-2/-3 incorporation when conserved amino acids within the N-terminal tail are mutated. We are, at the present time, unsure as to the mechanism by which these amino acids are important for conjugation of SUMO-2/-3. Given the observation that this tail is necessary, but not sufficient, for the interaction of C/EBPβ-1 with the large, multisubunit Swi-Snf complex (17Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), it is possible that the amino acids are involved in creating a structural change of the entire transcription factor, which distinguishes C/EBPβ-1 from C/EBPβ-2. Alternatively, the N terminus could be bound by a "bridging" factor necessary for interaction with components of the Swi-Snf complex and for interaction with the SUMO conjugation machinery. Unfortunately, there are currently no structural studies of the entirety of C/EBPβ; studies examining the differences in the structure of these two activator proteins will prove interesting. Mutation of lysine 173 mostly ablates the incorporation of SUMO-2/-3 into C/EBPβ-1. This sequence matches the SUMO target consensus and is likely the major site of conjugation of SUMO-2/-3 as well as SUMO-1 (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). C/EBPβ-2, but not C/EBPβ-1, is able to activate the cyclin D1 promoter despite the fact that both proteins are able to bind a consensus C/EBP site within the promoter (15Eaton E.M. Hanlon M. Bundy L. Sealy L. J. Cell. Physiol. 2001; 189: 91-105Crossref PubMed Scopus (63) Google Scholar). We extend this observation by demonstrating that lysine 173 is critical for the inability of C/EBPβ-1 to activate this promoter. Furthermore, we extend the observation made by Kim et al. (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), who used a Gal4-C/EBPβ activation domain fusion protein to show that the conserved lysine 134 in the rat was necessary for function of the repression domain. When the authors mutated the lysine to an alanine, they observed an increase in transactivation potential at a Gal4 binding site reporter construct (9Kim J. Cantwell C.A. Johnson P.F. Pfarr C.M. Williams S.C. J. Biol. Chem. 2002; 277: 38037-38044Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This is an expansion of earlier works, describing an increase in activation potential of a C/EBPβ-Gal4 fusion protein when certain domains (termed repression domains or conserved regions) were deleted (19Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. Genes Dev. 1994; 8: 2781-2791Crossref PubMed Scopus (210) Google Scholar, 20Williams S.C. Baer M. Dillner A.J. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar). Here, we show that mutation of this conserved lysine in the context of the entire protein disrupts the repression functions of C/EBPβ-1 at the cyclin D1 promoter. The apparent sumoylation of this lysine does not appear to influence subnuclear localization of C/EBPβ-1. Likely, sumoylation is affecting protein-protein interactions, such as with co-activators, co-repressors, or chromatin remodeling machinery, or blocking the transactivation potential via an intramolecular interaction. Recently, SUMO-dependent repression of the coactivator p300 function was shown to be mediated by recruitment of HDAC6 (21Girdwood D. Bumpass D. Vaughan O.A. Thain A. Anderson L. Snowden A.W. Garcia-Wilson E. Perkins N.D. Hay R.T. Mol. Cell. 2003; 11: 1043-1054Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). Whether histone deacetylases are involved in C/EBPβ-1 repression and whether their recruitment is SUMO-dependent will be interesting to determine in future studies. Although non-sumoylated C/EBPβ-1 (the lysine 173 mutant) behaves similarly to C/EBPβ-2 at the cyclin D1 promoter in a transient transfection assay, further studies will be necessary to determine whether non-sumoylated C/EBPβ-1 is functionally equivalent to C/EBPβ-2 at the entire set of genes that may be subject to C/EBPβ regulation.
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