The F-box Protein β-TrCp1/Fbw1a Interacts with p300 to Enhance β-Catenin Transcriptional Activity
2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês
10.1074/jbc.m901248200
ISSN1083-351X
AutoresErin A. Kimbrel, Andrew L. Kung,
Tópico(s)Cancer-related Molecular Pathways
ResumoHyperactivated β-catenin is a commonly found molecular abnormality in colon cancer, and its nuclear accumulation is thought to promote the expression of genes associated with cellular proliferation and transformation. The p300 transcriptional co-activator binds to β-catenin and facilitates transcription by recruiting chromatin remodeling complexes and general transcriptional apparatus. We have found that β-TrCp1/Fbw1a, a member of the Skp1/Cullin/Rbx1/F-box E3 ubiquitin ligase complex, binds directly to p300 and co-localizes with it to β-catenin target gene promoters. Our data show that Fbw1a, which normally targets β-catenin for degradation, works together with p300 to enhance the transcriptional activity of β-catenin, whereas other F-box/WD40 proteins do not. Fbw1a also cooperates with p300 to co-activate transcription by SMAD3, another Fbw1a ubiquitylation target, but not p53 or HIF-1α, which are substrates for other ubiquitin ligase complexes. These results suggest that, although Fbw1a is part of a negative feedback loop for controlling β-catenin levels in normal cells, its overexpression and binding to p300 may contribute to hyperactivated β-catenin transcriptional activity in colon cancer cells. Hyperactivated β-catenin is a commonly found molecular abnormality in colon cancer, and its nuclear accumulation is thought to promote the expression of genes associated with cellular proliferation and transformation. The p300 transcriptional co-activator binds to β-catenin and facilitates transcription by recruiting chromatin remodeling complexes and general transcriptional apparatus. We have found that β-TrCp1/Fbw1a, a member of the Skp1/Cullin/Rbx1/F-box E3 ubiquitin ligase complex, binds directly to p300 and co-localizes with it to β-catenin target gene promoters. Our data show that Fbw1a, which normally targets β-catenin for degradation, works together with p300 to enhance the transcriptional activity of β-catenin, whereas other F-box/WD40 proteins do not. Fbw1a also cooperates with p300 to co-activate transcription by SMAD3, another Fbw1a ubiquitylation target, but not p53 or HIF-1α, which are substrates for other ubiquitin ligase complexes. These results suggest that, although Fbw1a is part of a negative feedback loop for controlling β-catenin levels in normal cells, its overexpression and binding to p300 may contribute to hyperactivated β-catenin transcriptional activity in colon cancer cells. It is estimated that over 140,000 new cases of colorectal cancer were diagnosed in the United States in 2008. 2American Cancer Society (2008) Overview: Colon and Rectum Cancer, available on the web. On a molecular level, increased activity of the Wnt pathway is a common feature, with alteration(s) at various points in the pathway occurring in almost all cases of the disease and resulting in enhanced stability and nuclear accumulation of the β-catenin protein (2Fodde R. Smits R. Clevers H. Nat. Rev. Cancer. 2001; 1: 55-67Crossref PubMed Scopus (774) Google Scholar, 3Chen X. Yang J. Evans P.M. Liu C. Acta Biochim. Biophys. Sin. (Shanghai). 2008; 40: 577-594Crossref PubMed Scopus (39) Google Scholar). 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Likewise, the ubiquitin-conjugating enzyme Rsp5 and ubiquitin ligase Ubch7 have both been shown to independently serve as co-activators for steroid hormone receptors, the latter occurring through its interaction with the p160 co-activator, SRC1 (23Huibregtse J.M. Yang J.C. Beaudenon S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3656-3661Crossref PubMed Scopus (182) Google Scholar, 24Verma S. Ismail A. Gao X. Fu G. Li X. O'Malley B.W. Nawaz Z. Mol. Cell. Biol. 2004; 24: 8716-8726Crossref PubMed Scopus (78) Google Scholar). Additionally, an interaction between the yeast transcription factor, Gal4, and the ubiquitin ligase GCN4 was found to enhance Gal4-mediated gene activation (25Lipford J.R. Smith G.T. Chi Y. Deshaies R.J. Nature. 2005; 438: 113-116Crossref PubMed Scopus (159) Google Scholar). In this study, we found that p300 and CBP bind to β-TrCp1/Fbw1a, the substrate recognition partner associated with the Skp1/Cullin/Rbx1/F-box (SCF) E3 ubiquitin ligase complex that normally targets β-catenin for proteasome-mediated degradation (26Latres E. Chiaur D.S. Pagano M. Oncogene. 1999; 18: 849-854Crossref PubMed Scopus (377) Google Scholar). We found that p300/CBP, Fbw1a, and β-catenin co-localized to the promoters of target genes and that Fbw1a activated β-catenin-mediated transcription. Collectively, the data presented here provide support for the emerging theme that the integration of transcriptional activation complexes with ubiquitination machinery is a fundamental mechanism for regulating gene transcription. DNA Plasmids—The vector, pGEX6P.1 (Amersham Biosciences) was used to express p300 fragments fused to GST. CMV-p300-HA (Upstate Biotechnology) was the source of human p300 cDNA, and appropriate restriction enzymes were used for subcloning purposes. pcDNA3-β-TrCp1(Fbw1a)-FLAG. Fbw2-FLAG were kindly provided by Michele Pagano (New York University School of Medicine). pcDNA3-Fbw8-FLAG was kindly provided by William Kaelin (Dana-Farber Cancer Institute). pcDNA3-Parkin-FLAG was kindly provided by Ted Dawson (Johns Hopkins University). TOPflash/FOPflash luciferase reporters (pOT-luc and pOF-luc) were from Upstate Biotechnology. pGL4.14-cyclinD1-luciferase, containing 1882 bp of the human cyclin D1 promoter, was kindly provided by David Fisher (Dana-Farber Cancer Institute). β-Catenin and LEF expression vectors were gifts from Ramesh Shivdasani (Dana-Farber Cancer Institute). pCF-SMAD3 was a gift from Toshi Shioda (Massachusetts General Hospital). CAGA(12)-luciferase was kindly provided by Baogiang Guo (University of Manchester, UK). p21-luciferase was created by PCR amplification of a 1-kb promoter fragment and subcloning the fragment into the pGL3-basic vector (Promega). The 3xHRE-luciferase vector has been previously described (27Kung A.L. Zabludoff S.D. France D.S. Freedman S.J. Tanner E.A. Vieira A. Cornell-Kennon S. Lee J. Wang B. Wang J. Memmert K. Naegeli H.U. Petersen F. Eck M.J. Bair K.W. Wood A.W. Livingston D.M. Cancer Cell. 2004; 6: 33-43Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Phage Display Screening—p300 N-terminal and C-terminal fragments (residues 1–595 and 1929–2261) and CBP N-terminal and C-terminal fragments (1–615 and 1970–2292) were fused to GST, expressed in BL21 bacteria, and purified on glutathione beads (Amersham Biosciences) according to the manufacturer's instructions. The four p300 and CBP fragments were cleaved from the GST moiety with Precision Protease (Amersham Biosciences). The cleaved fragments were then chemically coupled to Dynal M270 carboxylic acid magnetic beads, according to the manufacturer's instructions. Target proteins, immobilized on beads, were blocked with 1 mg/ml bovine serum albumin in PBS. 10 μl of the M13 phage library, (PhD-7, New England Biolabs) diluted in 100 μl of TBS plus 0.1% Tween 20 (TBST) was added to each of the 4 samples for 1 h at room temperature, rotating tubes end over end. Samples were then washed 10 times with TBST, and bound phages were eluted with 0.2 m glycine, pH 2.2, for 10 min at room temperature. Eluted phage were neutralized with Tris-HCl (pH9.1), amplified for 5 h, and titered. 2 × 1010 amplified phage from round 1 panning were used as input for round 2 panning for each sample. Three total rounds of panning were performed as in round 1, except that the concentration of Tween in the TBST was increased from 0.1% to 0.5% in rounds 2 and 3. To increase the diversity of recovered peptides, individual phage from the second round were plaque-purified for sequencing with an M13 reverse primer (New England Biolabs). In Vitro Interaction Assays—The proteins EGFP, EGFP-peptide, or Fbw1a-FLAG were produced by linked in vitro transcription-translation (TnT, Promega, Madison, WI). Mutated Fbw1a, in which residues 493–497 (KVWDL) were replaced by NERDR, was created with the QuikChange site-directed mutagenesis kit (Stratagene) and then in vitro transcribed and translated. Binding to GST fusion proteins, on glutathione beads or to fragments coupled to magnetic beads was performed in NETN-A (50 mm NaCl, 1 μm EDTA, 20 mm Tris, pH 8.0, 0.5% Nonidet P-40, 10 μm ZnSO4) overnight at 4 °C. Six washes were performed with NETN-B (200 mm NaCl, 1 μm EDTA, 20 mm Tris, pH 8.0, 0.5% Nonidet P-40, 10 μm ZnSO4). Samples were then subjected to SDS-PAGE and transferred to nitrocellulose for Western blotting with anti-EGFP antibody (Clontech), anti-FLAG antibody (Sigma), anti-HIF1α antibody (Novus Biologics), or anti-SRC1 (Neomarkers). Cell Culture, Coimmunoprecipitation, and Western Blotting— All cell lines used (HCT116, 293T, and HeLa) were grown in Dulbecco's modified Eagle's medium plus 10% fetal calf serum, in a 37 °C, 10% CO2 incubator. Transient transfections were performed with FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Whole cell lysates were made in radioimmune precipitation assay buffer containing protease inhibitors (Complete, Roche Applied Science). Nuclear extracts were made by incubating cells in a hypotonic buffer (10 mm Tris, pH 7.6, 10 mm KCl, 1.5 mm MgCl2), homogenizing with a glass tissue grinder, and centrifuging to isolate nuclear pellets. Nuclear pellets were then placed in a low salt (20 mm KCl) buffer and slowly moved to a high salt buffer (1.2 m KCl) to extract nuclear protein. Dialysis in BC100 buffer (200 mm Tris, 100 mm KCl, 10% glycerol) was performed to allow buffer exchange. Co-immunoprecipitation was performed for 4 h to overnight at 4 °C with the indicated antibodies. Protein A/G beads, mouse IgG, and rabbit IgG were from Santa Cruz Biotechnology. For endogenous co-immunoprecipitation, the anti-p300 antibody RW128 (Upstate Biotechnology) was conjugated to protein G beads with the Seize X Protein G IP kit (Pierce) according to the manufacturer's instructions. Washes were performed with radioimmune precipitation assay buffer or PBST (PBS plus 0.5% Triton X-100). Samples were placed in SDS-containing loading dye, subjected to SDS-PAGE, and transferred to nitrocellulose for immunoblotting for Fbw1a (Invitrogen and Santa Cruz Biotechnology), Gal4DBD (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and the HA tag (Upstate and Covance). Chromatin Immunoprecipitation (ChIP)—Lithium Chloride was from Sigma. Recombinant Wnt3a was from R&D Systems. ChIP assays were performed with a ChIP kit (Millipore) according to the manufacturer's instructions. Briefly, proteins/DNA were cross-linked with 1% formaldehyde, cells were lysed, and chromatin was sheared with sonication. After centrifugation, lysates were diluted and proteins were immunoprecipitated overnight with antibodies against β-catenin (Santa Cruz Biotechnology), p300 (Santa Cruz Biotechnology), Fbw1a (Invitrogen and Santa Cruz Biotechnology), or FLAG (Sigma). Samples were washed, eluted, and the cross-links were reversed at 65 °C for 4 h. The isolated DNA was purified using a PCR purification kit (Qiagen). Real-time PCR was performed on a Stratagene MX3000P, using Quantitect Sybr green master mix (Qiagen). ChIP-reChIP was performed in the same manner as above, expect that eluates from the primary β-catenin ChIP were rediluted 10-fold and re-immunoprecipitated with anti-p300 and anti-Fbw1a antibodies prior to reversal of cross-links. As previously reported (28Chen Y.H. Yang C.K. Xia M. Ou C.Y. Stallcup M.R. Nucleic Acids Res. 2007; 35: 2084-2092Crossref PubMed Scopus (21) Google Scholar), cyclin D1 promoter primers were: fwd (5′-CGG GGC AGC AGA AGC GAG A-3′) and rev (5′-GTG AGT AGC AAA GAA ACG TGG-3′); cyclin D1 control primers (3892 bp upstream of promoter) were: fwd (5′-GGT CCT CCC CGC AGT CTT C-3′) and rev (5′-CTC TCC CCC GCA GTC AGG-3′). As previously reported (29Li J. Wang C.Y. Nat. Cell Biol. 2008; 10: 160-169Crossref PubMed Scopus (137) Google Scholar), axin2 promoter primers were: fwd (5′-CTG GAG CCG GCT GCG CTT TGA TAA-3′) and rev (5′-CGG CCC CGA AAT CCA TCG CTC TGA), and axin2 control primers (2542 bp downstream from ATG start site, within open reading frame) were: fwd (5′-CTG GCT TTG GTG AAC TGT TG-3′) and rev (5′-AGT TGC TCA CAG CCA AGA CA-3′). Enolase1 primers were: fwd (5′-TGT AGT GGT GCG GGC GAA ACT CTG-3′) and rev (5′-AGA GCG ACG CTG AGT GCG T-3′). Luciferase Assays and Fbw1a Knockdown—Cells were seeded in 24-well plates and transfections were performed in triplicate wells. A total of 3 μg of DNA per triplicate was transfected into cells with FuGENE 6 according to the manufacturer's instructions. Depending on the specific experiment, 0.7–1.2 μg of firefly luciferase reporter, 0.05–0.2 μg of Renilla luciferase, and between 1.2 and 1.8 μg of CMV-promoter vectors were used per triplicate, per experiment. The total amount of CMV-based vectors (pcDNA3.1 empty vector plus pcDNA3.1-Fbox-encoding) was always held constant in each experiment. After balancing for CMV levels, the total amount of DNA in each transfection was brought up to 3 μg per triplicate by using pBS2. 48 h after transfection, cells were lysed and luciferase assays were performed with a dual luciferase assay system (Promega). Activities of the firefly luciferase reporters were normalized to that of the internal control vector, pCMV-Renilla-luciferase. A Veritas microplate luminometer (Turner Biosystems) was used to collect luciferase readings. Knockdown of Fbw1a was performed with a combination of two lentiviral short hairpin (sh) RNA constructs from OpenBiosystems and one Dharmacon siRNA sequence that had been adapted for shRNA lentiviral infection. Briefly, 293T cells were transfected with lentiviral packaging vectors and a vector containing either a non-silencing or shRNA sequence directed against Fbw1a. 48 and 72 h after transfection, equal volumes of filtered supernatants were added to 293T cells for infection with either non-silencing or Fbw1a knockdown viruses. Puromycin (2 μg/ml) was used for selection and fluorescence from the GFP-containing shRNA vectors was used to confirm nearly 100% infection efficiency. 96–120 h after infection, quantitative reverse transcription-PCR and immunoblotting were performed to confirm Fbw1a knockdown. RNA was extracted using TRIzol and mRNA levels were normalized to GAPDH after quantitative reverse transcription-PCR. Primer sequences for Fbw1a mRNA were fwd (5′-GCT GAA CTT GTG TGC AAG GA-3′) and rev (5′-TAC TGT CCC CAT CCT CTT CG-3′); axin2 mRNA, fwd (5′-AGT GTG AGG TCC ACG GAA AC-3′) and rev (5′-CTT CAC ACT GCG ATG CAT TT); cyclinD1 mRNA, fwd (5′-GCG GAG GAG AAC AAA CAG AT-3′) and rev (5′-TGA GGC GGT AGT AGG ACA GG-3′). Quantification of immunoblot bands was performed using the Photoshop histogram function. Absolute band intensity was determined using the mean and pixel values for each band; relative band intensity was determined by normalizing to GAPDH. p300 Binds Peptides with Homology to F-box/WD40 Proteins— To identify peptides that target proteins for binding to p300 and CBP, we used fragments of p300 and CBP to screen a random 7-amino acid M13 phage-displayed peptide library. Four protein fragments were used as targets for the screen, two from p300 and the corresponding two from CBP (Fig. 1A). The N-terminal fragment of p300 encompassing amino acids 1–595 (and corresponding CBP fragment) includes the N terminus, CH1, and part of the KIX domains that are known binding sites for nuclear receptors, hypoxia-inducible factor 1 α (HIF1α), and p53, among other transcription factors (8Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577Crossref PubMed Google Scholar, 30Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1283) Google Scholar, 31Jenster G. Spencer T.E. Burcin M.M. Tsai S.Y. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7879-7884Crossref PubMed Scopus (233) Google Scholar, 32De Guzman R.N. Liu H.Y. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 303: 243-253Crossref PubMed Scopus (100) Google Scholar, 33De Guzman R.N. Martinez-Yamout M.A. Dyson H.J. Wright P.E. J. Biol. Chem. 2004; 279: 3042-3049Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The C-terminal fragment (p300 amino acids 1929–2261 or corresponding CBP fragment) includes the SID/IBiD domain and its flanking sequences, which contain known binding sites for the SMAD proteins, p53, ETS2, E1A, and the p160 co-activators SRC1, GRIP1, and AIB1 (34Teufel D.P. Freund S.M. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7009-7014Crossref PubMed Scopus (157) Google Scholar, 35Matsuda S. Harries J.C. Viskaduraki M. Troke P.J. Kindle K.B. Ryan C. Heery D.M. J. Biol. Chem. 2004; 279: 14055-14064Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 36Livengood J.A. Scoggin K.E. Van Orden K. McBryant S.J. Edayathumangalam R.S. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 9054-9061Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 37Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-L. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar). We did not include the CH3 domain within the C-terminal fragment, because CH3 and CH1 are structurally highly similar and several transcription factors bind redundantly to these two domains (32De Guzman R.N. Liu H.Y. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 303: 243-253Crossref PubMed Scopus (100) Google Scholar). After GST purification, the fragments were cleaved of their GST moieties (to avoid nonspecific peptide binding) and chemically coupled to magnetic beads for in solution library screening. In vitro interaction assays confirmed that coupling to magnetic beads did not alter the structure of the target proteins. As expected, immobilization of the N-terminal p300 and CBP fragments to magnetic beads conferred binding to in vitro translated HIF1α and coupling of the C-terminal p300 and CBP fragments to beads conferred binding to SRC1 (supplemental Fig. S1). In solution library screening was performed with an M13 phage library whose complexity of ∼1011 different peptides encompasses all possible permutations of peptides 7 amino acids in length. Three successive rounds of panning were performed to enrich for p300/CBP-binding peptides, however, to provide for diversity of recovered peptides, phage from the second round of panning were plaque-purified for sequencing. 6 of the 24 peptides sequenced from the screen of N-terminal p300 and CBP fragments contained the motif K(V/L)WXL (Fig. 1B), whereas none of the 27 peptides sequenced from the screen of C-terminal fragments contained it (supplemental Fig. S1 B), indicating a significant enrichment for binding of this motif to the N-terminal fragments (p = 0.007, Fisher's exact test). Although other motifs emerged in the collection of sequenced peptides (supplemental Fig. S1B), none of these occurred as often as K(V/L)WXL. Of note, the proline-rich motif P(L/R)XXP appeared 3–4 times in the C-terminal fragment screen. These peptides are very similar to the previously characterized proline-repeat motifs in the p53 transactivation domain, which are known to bind p300/CBP and are important for DNA-dependent acetylation of p53 (38Dornan D.S.H. Burch L. Smith A.J. Hupp T.R. Mol. Cell. Biol. 2003; 23: 8846-8861Crossref PubMed Scopus (80) Google Scholar). Given the prominence of the K(V/L)WXL motif in our collection of peptides from the N-terminal fragment screen, we chose to focus on elucidating the significance of this motif. To verify that the K(V/L)WXL peptide was sufficient to direct binding to p300 and CBP, we cloned one of the identified peptides (KVWTLNY) (Fig. 1B) into a solution-exposed loop of the enhanced green fluorescent protein (EGFP). In vitro interaction assays demonstrated that insertion of this peptide conferred binding of EGFP to the N-terminal fragments of both p300 and CBP, as well as a smaller fragment containing only the CH1 domain of p300 (Fig. 1, C and D). We searched the proteomics data base at ScanProsite to determine whether any endogenous proteins contain this binding motif. We found that several members of the F-box protein family contain a K(V/L)WXL motif (Fig. 2A). F-box proteins serve as one of the four subunits in the Skp1/Cullin/Rbx/F-box (SCF) complex, which functions as an E3 ubiquitin ligase. F-box proteins are further subdivided based on the type of their substrate recognition domain, either a WD40 domain, leucine-rich region, or F-box only with no defined substrate recognition region. In our data base search, all but one of the identified F-box proteins were F-box/WD40 (Fbw) proteins, whereas one F-box only (Fbx18) protein also had the motif. None of the F-box/leucine-rich repeat (Fbl) family members contained a K(V/L)WXL motif. WD40 repeat domains fold into a seven-blade propeller, and the crystal structure of Fbw1a (39Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Mol. Cell. 2003; 11: 1445-1456Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar) reveals that the K(V/L)WXL motif resides within the sixth blade, in a location likely accessible for protein-protein interactions. p300 and Fbw1a Interact in Vitro and in Cells—To determine if p300 interacts with Fbw proteins, we performed an in vitro interaction assay, which demonstrated that full-length Fbw1a bound to the N-terminal fragment of p300 and to the smaller CH1 domain located within this fragment (Fig. 2B). Interestingly, Fbw1a also bound to a C-terminal region of p300 that includes the CH3 domain but did not bind to a middle region of p300 that includes the CH2/HAT domains (Fig. 2C). CH1 and CH3 are highly similar in both primary sequence and structure (32De Guzman R.N. Liu H.Y. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 303: 243-253Crossref PubMed Scopus (100) Google Scholar) and several transcription factors such as p53, E1A, and Ets-1 bind to both domains. To determine if the K(V/L)WXL motif is necessary for Fbw1a binding to p300, we mutated the KVWDL sequence within Fbw1a to NERDR. This mutation abolished binding to the N-terminal and CH1 p300 fragments, but it did not abrogate binding to the CH3-to-end fragment (Fig. 2D). This suggests multiple interaction surfaces exist between Fbw1a and p300, similar to those for p53 (34Teufel D.P. Freund S.M. Bycroft M. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7009-7014Crossref PubMed Scopus (157) Google Scholar). The Fbw protein, Fbw8, which does not contain a K(V/L)WXL motif, was able to bind to the CH3-to-end fragment but had little or no binding to the N-terminal or CH1 fragments (Fig. 2D). Collectively, these results show that the K(V/L)WXL motif in Fbw1a can direct binding to the N-terminal/CH1 domain of p300, but, like certain transcription factors, multiple interaction surfaces mediate the binding between these two classes of proteins. Next, we wanted to characterize the p300/Fbw interaction within mammalian cells. 293T cells were transiently transfected with HA-tagged p300 alone or together with FLAG-tagged Fbw1a, Fbw2, Parkin (a Fbl protein without the K(V/L)WXL motif), or Fbw8 (40Chiaur D.S. Murthy S. Cenciarelli C. Parks W. Loda M. Inghirami G. Demetrick D. Pagano M. Cytogenet. Cell Genet. 2000; 88: 255-258Crossref PubMed Scopus (25) Google Scholar, 41Tanaka K. Suzuki T. Hattori N. Mizuno Y. Biochim. Biophys. Acta. 2004; 1695: 235-247Crossref PubMed Scopus (102) Google Scholar, 42Chung K.K. Thomas B. Li X. Pletnikova O. Troncoso J.C. Marsh L. Dawson V.L. Dawson T.M. Science. 2004; 304: 1328-1331Crossref PubMed Scopus (675) Google Scholar). Forty-eight hours after transfection, immunoprecipitation with an anti-FLAG antibody was performed. Western blotting revealed that HA-p300 was readily co-immunoprecipitated in cells expressing FLAG-tagged Fbw1a, Fbw2, and Fbw8 but not from control cells or cells expressing Parkin-FLAG (Fig. 3A). We also wanted to determine if endogenous p300 and Fbw1a could interact. HeLa cell nuclear extracts were prepared, and p300 was immunoprecipitated. Western blotting revealed that Fbw1a was co-immunoprecipitated with p300 but not with a control IgG/mock IP (Fig. 3B). These results verify the binding of Fbw1a to p300 within cells. To define the region(s) of Fbw1a that can interact with p300, we expressed p300-HA and three Gal4DBD-tagged Fbw1a fragments in 293T cells (Fig. 4A). Co-immunoprecipitation and Western blotting verified that the WD40 domain, which contains the K(V/L)WXL motif, confers binding to p300, whereas other fragments of Fbw1a (F-box, mid) did not (Fig. 4B). This result, along with the in vitro interaction studies (Figs. 1 and 2), demonstrates that the WD40 domain is necessary and sufficient to target Fbw1a for binding to p300. To confirm the binding surfaces on p300 that are able to bind Fbw1a in cells, six HA-tagged p300 fragments (Fig. 5A) were co-transfected into 293T cells with Fbw1a-FLAG. Immunoprecipitation/Western blotting (
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