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BTB Domain-containing Speckle-type POZ Protein (SPOP) Serves as an Adaptor of Daxx for Ubiquitination by Cul3-based Ubiquitin Ligase

2006; Elsevier BV; Volume: 281; Issue: 18 Linguagem: Inglês

10.1074/jbc.m600204200

ISSN

1083-351X

Autores

Jeong Eun Kwon, Muhnho La, Kyu Hee Oh, Young Mi Oh, Gi Ryang Kim, Jae Hong Seol, Sung Hee Baek, Tomoki Chiba, Keiji Tanaka, Ok Sun Bang, Cheol O. Joe, Chin Ha Chung,

Tópico(s)

Cell death mechanisms and regulation

Resumo

Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis. Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis. Ubiquitin-dependent proteolysis plays an essential role in the regulation of a variety of cellular processes, including cell proliferation, differentiation, and apoptosis (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6793) Google Scholar, 2Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1452) Google Scholar, 3King R.W. Deshaies R.J. Peters J.M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar). Ubiquitin (Ub) 3The abbreviations used are: Ub, ubiquitin; E3, Ub-ligating enzyme; HA, hemagglutinin; PBS, phosphate-buffered saline; shRNA, short hairpin RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling; PARP, poly(ADP-ribose) polymerase. 3The abbreviations used are: Ub, ubiquitin; E3, Ub-ligating enzyme; HA, hemagglutinin; PBS, phosphate-buffered saline; shRNA, short hairpin RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling; PARP, poly(ADP-ribose) polymerase. is covalently attached to target proteins by a cascade enzyme system consisting of Ub-activating (E1), conjugating (E2), and ligating (E3) enzymes (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6793) Google Scholar, 4Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (731) Google Scholar). Ub E3 ligases that confer the substrate specificity have been grouped into two families; the HECT-domain family that is defined by its homology to E6-associated protein (E6AP) and the RING family carrying RING-finger domain that is essential for the Ub ligase activity (5Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar, 6Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar). One of the well defined RING E3 ligases is the Skp1/Cul1/F-box protein complex, in which Cul1 serves as a scaffold molecule that interacts with Skp1 and a small RING-finger protein Roc1, also known as Hrt1 and Rbx1 (7Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin Jr., W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (662) Google Scholar, 8Ohta T. Michel J.J. Schottelius A.J. Xiong Y. Mol. Cell. 1999; 3: 535-541Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 9Zheng N. Schulman B.A. Song L. Miller J.J. Jeffrey P.D. Wang P. Chu C. Koepp D.M. Elledge S.J. Pagano M. Conaway R.C. Conaway J.W. Harper J.W. Pavletich N.P. Nature. 2002; 416: 703-709Crossref PubMed Scopus (1151) Google Scholar). F-box proteins are recruited to the complex by binding to the Skp1 adaptor protein.At least six Cul members have been identified: Cul1, Cul2, Cul3, Cul4A, Cul4B, and Cul5 (10Kipreos E.T. Lander L.E. Wing J.P. He W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Of these, Cul3 is known to mediate the degradation of several proteins, such as cyclin E (11Singer J.D. Gurian-West M. Clurman B. Roberts J.M. Genes Dev. 1999; 13: 2375-2387Crossref PubMed Scopus (330) Google Scholar), but the molecular composition of Cul3-based Ub ligase was unknown. Recently, a large family of proteins having BTB (Bric-a-brac/Tramtrack/Broad complex) domain has been identified as novel Cul3-interacting proteins (12Furukawa M. He Y.J. Borchers C. Xiong Y. Nat. Cell Biol. 2003; 5: 1001-1007Crossref PubMed Scopus (332) Google Scholar). Most BTB proteins, but not all, have additional domains for protein-protein interaction, such as zinc fingers, Kelch repeats, and MATH motifs. Furthermore, a subset of proteins containing BTB domain has been identified to function as substrate-specific adaptors that bind to Cul3. Specifically, MEL-26, a homolog of human SPOP (speckle-type POZ protein) in Caenorhabditis elegans, was first identified as a BTB protein that serves as a specific adaptor of MEI-1 for the ubiquitination by Cul3-based Ub ligase and subsequent degradation by the proteasome (13Xu L. Wei Y. Reboul J. Vaglio P. Shin T.H. Vidal M. Elledge S.J. Harper J.W. Nature. 2003; 425: 316-321Crossref PubMed Scopus (383) Google Scholar). MEI-1 is a subunit of the katanin-like microtubule severing heterodimer MEI-1/MEI-2 that localizes to the spindles and the chromosomes during meiosis (14Srayko M. Buster D.W. Bazirgan O.A. McNally F.J. Mains P.E. Genes Dev. 2000; 14: 1072-1084PubMed Google Scholar). SPOP BTB protein has also been shown to mediate the ubiquitination of the Polycomb group BMI and the variant histone MacroH2A (15Hernandez-Munoz I. Lund A.H. van der Stoop P. Boutsma E. Muijrers I. Verhoeven E. Nusinow D.A. Panning B. Marahrens Y. van Lohuizen M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7635-7640Crossref PubMed Scopus (246) Google Scholar). In addition, Keap1 BTB protein was shown to recruit Nrf2 to Cul3 (16Cullinan S.B. Gordan J.D. Jin J. Harper J.W. Diehl J.A. Mol. Cell. Biol. 2004; 24: 8477-8486Crossref PubMed Scopus (750) Google Scholar, 17Furukawa M. Xiong Y. Mol. Cell. Biol. 2005; 25: 162-171Crossref PubMed Scopus (573) Google Scholar, 18Kobayashi A. Kang M.I. Okawa H. Ohtsuji M. Zenke Y. Chiba T. Igarashi K. Yamamoto M. Mol. Cell. Biol. 2004; 24: 7130-7139Crossref PubMed Scopus (1595) Google Scholar, 19Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell. Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (946) Google Scholar). Nrf2 is a transcription factor that regulates the expression of anti-oxidant genes upon oxidative stress. In Schizosaccharomyces pombe, Btb1p, Btb2p, and Btb3p interact with Cul3, but their functions remain unknown (20Geyer R. Wee S. Anderson S. Yates J. Wolf D.A. Mol. Cell. 2003; 12: 783-790Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Therefore, so far only a few protein substrates have been shown to interact with BTB proteins for their ubiquitination by Cul3-based Ub ligases.Daxx was originally identified as a protein that binds to the death domain of Fas receptor by yeast two-hybrid screening (21Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). Daxx interacts with the apoptosis signal-regulating kinase 1 (ASK1) and promotes Fas-mediated apoptosis through the activation of Jun N-terminal kinase (22Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (527) Google Scholar). Subsequent studies have also shown that Daxx behaves as a pro-apoptotic protein under various stress conditions (21Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar, 23Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell Biol. 2001; 3: 708-714Crossref PubMed Scopus (301) Google Scholar, 24Torii S. Egan D.A. Evans R.A. Reed J.C. EMBO J. 1999; 18: 6037-6049Crossref PubMed Scopus (235) Google Scholar, 25Zhong S. Salomoni P. Ronchetti S. Guo A. Ruggero D. Pandolfi P.P. J. Exp. Med. 2000; 191: 631-640Crossref PubMed Scopus (193) Google Scholar). On the contrary, homozygous deletion of the Daxx gene has been shown to cause extensive apoptosis and embryonic lethality, suggesting that Daxx is an anti-apoptotic protein (26Michaelson J.S. Bader D. Kuo F. Kozak C. Leder P. Genes Dev. 1999; 13: 1918-1923Crossref PubMed Scopus (193) Google Scholar). Recent studies also support that Daxx is anti-apoptotic rather than pro-apoptotic (27Cermak L. Simova S. Pintzas A. Horejsi V. Andera L. J. Biol. Chem. 2002; 277: 7955-7961Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 28Chen L.Y. Chen J.D. Mol. Cell. Biol. 2003; 23: 7108-7121Crossref PubMed Scopus (97) Google Scholar, 29Wu S. Loke H.N. Rehemtulla A. Neoplasia. 2002; 4: 486-492Crossref PubMed Scopus (38) Google Scholar).Daxx also plays a role as a transcription regulator by interacting with various nuclear proteins involved in transcription. Daxx interacts with HDAC-II, core histones, and a chromatin-associated protein, Dek, suggesting that Daxx represses basal transcription through chromatin remodeling (30Hollenbach A.D. McPherson C.J. Mientjes E.J. Iyengar R. Grosveld G. J. Cell Sci. 2002; 115: 3319-3330Crossref PubMed Google Scholar). Daxx has also been shown to form a ternary complex with homeodomain proteins Pax-3 and Pax-7 to inhibit transcription, whereas Pax-3/forkhead in human rhabdomyosarcoma (FKHR) fusion protein in alveolar rhabdomyosarcoma cells is resistant to Daxx inhibition (31Hollenbach A.D. Sublett J.E. McPherson C.J. Grosveld G. EMBO J. 1999; 18: 3702-3711Crossref PubMed Scopus (185) Google Scholar, 32Lehembre F. Muller S. Pandolfi P.P. Dejean A. Oncogene. 2001; 20: 1-9Crossref PubMed Scopus (93) Google Scholar). Similarly, Daxx interacts with ETS1 to repress transcriptional activation of the MMP1 gene (33Li R. Pei H. Watson D.K. Papas T.S. Oncogene. 2000; 19: 745-753Crossref PubMed Scopus (158) Google Scholar). It has also been demonstrated that Daxx suppresses cell death induced by p53 overexpression and p53-dependent stress response, thus acting as a negative regulator of p53 (34Zhao L.Y. Liu J. Sidhu G.S. Niu Y. Liu Y. Wang R. Liao D. J. Biol. Chem. 2004; 279: 50566-50579Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Interestingly, the level of Daxx protein has been shown to decrease in HeLa cells upon induction of apoptosis by actinomycin D or exposure to UV (28Chen L.Y. Chen J.D. Mol. Cell. Biol. 2003; 23: 7108-7121Crossref PubMed Scopus (97) Google Scholar). Moreover, Daxx has recently been shown to interact with SPOP (35La M. Kim K. Park J. Won J. Lee J.H. Fu Y.M. Meadows G.G. Joe C.O. Biochem. Biophys. Res. Commun. 2004; 320: 760-765Crossref PubMed Scopus (16) Google Scholar). These reports suggest a possibility that the functions of Daxx can be regulated by proteolysis.In the present study we demonstrate that SPOP recruits Daxx to Cul3 and promotes the ubiquitination of Daxx and subsequent degradation by the proteasome. Moreover, SPOP/Cul3-mediated degradation of Daxx reversed Daxx-mediated repression of ETS1- and p53-dependent transcription. We also demonstrate that Daxx degradation triggers apoptosis. Taken together, we suggest that SPOP/Cul3-Ub E3 ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis.EXPERIMENTAL PROCEDURESPlasmids and Cells—pGL3-MMP1-Luc and various vectors expressing Daxx, p53, ETS1, and wild-type and mutant forms of human SPOP were prepared as described (35La M. Kim K. Park J. Won J. Lee J.H. Fu Y.M. Meadows G.G. Joe C.O. Biochem. Biophys. Res. Commun. 2004; 320: 760-765Crossref PubMed Scopus (16) Google Scholar). SPOP cDNA was cloned into pEYFP-C1 (BD Biosciences Clontech) and pcDNA3.1-Myc-His (Invitrogen). The cDNAs for SPOP deletion fragments were cloned into p3XFLAG-CMV10 (Sigma). The cDNAs for human Roc1 and Culs were cloned into pcDNA3-FLAG, and Ub cDNA was cloned into pcDNA4-HisMax (Invitrogen). To prevent Nedd8 modification of Cul3, Lys-711 was replaced by Ser using the QuikChange site-directed mutagenesis kit (Stratagene). The Tyr-62 residue of Cul3 was also replaced by Gly to prevent its interaction with SPOP. Mutagenesis was performed using pcDNA3-FLAG-Cul3 as the template and the following mutagenic primers: Y62G, 5′-G GAG CTC TAT AGA AAT GCA GGT ACA ATG GTT TTG CAT AAA CAT GG-3′ (forward) and 5′-CC ATG TTT ATG CAA AAC CAT TGT ACC TGC ATT TCT ATA GAG CTC C-3′ (reverse); K711S, 5′-GCT ATA GTG CGG ATA ATG CGA TCT AGA AAG AAG ATG CAG C-3′ (forward) and 5′-G CTG CAT CTT CTT TCT AGA TCG CAT TAT CCG CAC TAT AGC-3′ (reverse). 293T, HeLa, and COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C under a humidified atmosphere. Transient transfections were performed using Lipofectamine Plus (Invitrogen) according to the manufacturer's instruction.Immunoprecipitation and Immunoblot—For immunoprecipitation, cells were lysed in 20 mm Tris-HCl (pH 7.4), 5 mm EDTA, 10 mm Na2P2O7, 10 mm NaF, 2 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and 1× protease inhibitor mixture (Roche Applied Science). Cell lysates were incubated with the appropriate antibodies for 2 h at 4°C and then with 50 μl of 50% slurry of protein A/G-Sepharose for the next hour. Bound proteins were eluted by boiling and subjected to SDS-PAGE followed by immunoblot with mouse anti-HA antibody (Roche Applied Science), rabbit anti-HA-antibody (Upstate Biotechnology, Inc.), mouse anti-FLAG M2 monoclonal antibody (Sigma), anti-Xpress antibody (Invitrogen), rabbit anti-Daxx-antibody (Calbiochem or Upstate), rabbit anti-Cul3-antibody (Zymed Laboratories Inc.), or anti-SPOP antiserum. Rabbit polyclonal anti-SPOP antiserum was raised against a recombinant C-terminal fragment of SPOP (amino acids 162-375). For in vitro interaction assay, HA-Daxx, MycHis-SPOP, and FLAG-Cul3 were in vitro translated using [35S]Met and TNT quick-coupled reticulocyte lysate system (Promega). 35S-Labeled proteins (20 μl) were incubated for 2 h with anti-HA antibody and then pulled down by treatment with protein A/G-Sepharose beads. Precipitates were subjected to SDS-PAGE followed by autoradiography.Immunocytochemistry—HeLa cells plated on gelatin-coated cover-glasses were fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature followed by permeabilization with 0.5% Triton X-100 in PBS. All subsequent dilutions and washes were carried out with PBS containing 0.1% Triton X-100. Nonspecific binding sites were saturated by incubation with a blocking solution (10% goat serum, 1% bovine serum albumin, and 1% gelatin in PBS) for 30 min. Cells were incubated with primary antibody for 1 h and washed with PBS containing 0.1% Triton X-100 four times with 10-min intervals. They were then incubated with fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated secondary antibody for 1 h and washed 4 times with 10-min intervals. 4,6-Diamidino-2-phenylindole was used for counter-staining the nuclei. The cover-glasses were mounted in Vectashield (Vector Laboratories, Inc.), and cells were observed under a Zeiss Axioplan II microscope or Image Restoration Microscope (Applied Precision).Transcription Assay—For assaying the transcription of ETS1-dependent luciferase reporter gene, 293T cells were cultured on 24-well plates and transiently transfected with pGL3-MMP1-Luc and various combinations of expression vectors for HA-Daxx, FLAG-ETS1, FLAG-SPOP, FLAG-Cul3, and FLAG-Roc1. For assaying the transcription of p53-dependent luciferase reporter gene, PG13-Luc was used as a reporter vector. The luciferase (Luc) activity was then determined using a luciferase assay kit (Promega). Total amounts of transfected vector DNAs were kept constant by supplementing the appropriate amount of empty pcDNA vector. The luciferase activity was then normalized relative to the co-expressed β-galactosidase activity.Short Hairpin RNAs (shRNA) Construction and Reverse Transcription-PCR—SPOP-specific shRNAs were synthesized and cloned into pSi-lencer 3.0 vector driven by the human H1 promoter (Ambion) with the following target sequences: 1, sense, 5′-GAT CCG TTC CAG GCT CAC AAG GCT ATC TTC AAG AGA GAT AGC CTT GTG AGC CTG GAA ATT TTT TGG AAA-3′, and antisense, 5′-AGC TTT CCA AAA AAT TCC AGG CTC ACA AGG CTA TCT CTC TTG AAG ATA GCC TTG TGA GCC TGG AAC G-3′; 2, sense, 5′-G ATC CGC TAT CAT GCT TCG GAT GTC TTC AAG AGA GAC ATC CGA AGC ATG ATA GTT TTT TGG AAA-3′, and antisense, 5′-AG CTT TTC CAA AAA ACT ATC ATG CTT CGG ATG TCT CTC TTG AAG ACA TCC GAA GCA TGA TAG CG-3′. To knock down SPOP mRNA, HeLa cells were transfected with shRNA vectors or a negative control vector (shControl). Total RNAs were prepared from cells by extracting with Trizol (Invitrogen), and subjected to reverse transcription-PCR. Reverse transcription reactions were performed using Superscript III reverse transcriptase (Invitrogen) and oligo(dT) primer by following the manufacturer's instruction. SPOP mRNA was amplified with primers against the unique region of SPOP (5′-GCC CTG GAG CGC TTA AAG G-3′ and 5′-GGA TTG CTT CAG GCG TTT GC-3′), and β-actin mRNA was amplified as an expression control.Terminal Deoxynucleotidyltransferase-mediated dUTP-fluorescein Nick End-labeling (TUNEL) Assay—HeLa cells cultured on cover-glasses were transfected with various combinations of expression vectors for SPOP, Cul3, Roc1, and Daxx. After incubation for 24 h, cells were fixed with 3.7% paraformaldehyde for 10 min and subjected to TUNEL assay by following the manufacturer's instruction (Roche Applied Science). The cover-glasses were mounted in Vectashield, and cells with positive TUNEL staining were counted under a Zeiss Axioplan II microscope. The nuclei were counterstained with 4,6-diamidino-2-phenylindole for counting total cell numbers.RESULTSInteraction of Human SPOP with Cul3—SPOP is a BTB protein that was initially identified as an auto-antigen of scleroderma patients (36Nagai Y. Kojima T. Muro Y. Hachiya T. Nishizawa Y. Wakabayashi T. Hagiwara M. FEBS Lett. 1997; 418: 23-26Crossref PubMed Scopus (95) Google Scholar). To define the sites within the human SPOP and Cul3 proteins for their interaction, we first generated Cul3 mutants based on the previous reports. The replacement of Tyr-50 by Gly of Pcu3p, a Cul3 homolog in S. pombe, prevents its ability to interact with BTB proteins (20Geyer R. Wee S. Anderson S. Yates J. Wolf D.A. Mol. Cell. 2003; 12: 783-790Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The replacement of Lys-711 by Ser in human Cul3 blocks its modification by Nedd8 (37Hori T. Osaka F. Chiba T. Miyamoto C. Okabayashi K. Shimbara N. Kato S. Tanaka K. Oncogene. 1999; 18: 6829-6834Crossref PubMed Scopus (243) Google Scholar, 38Ou C.Y. Lin Y.F. Chen Y.J. Chien C.T. Genes Dev. 2002; 16: 2403-2414Crossref PubMed Scopus (162) Google Scholar). Therefore, mutagenesis was performed to generate Y62G (Tyr-62 in Cul3 corresponds to Tyr-50 in Pcu3p) and K711S (Fig. 1A, upper panel). HA-SPOP was expressed in 293T cells with FLAG-Cul3 or its mutant forms. Cell lysates were prepared and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblot with anti-HA or anti-FLAG antibody. Wild-type Cul3, but not Y62G, was co-precipitated with SPOP (Fig. 1A, lower panel), indicating that Tyr-62 in human Cul3 is also essential for the interaction between Cul3 and SPOP. On the other hand, K711S could be co-precipitated with SPOP, indicating that the interaction between Cul3 and SPOP is independent of Nedd8 modification.SPOP has been shown to interact with Cul3 via BTB domain (12Furukawa M. He Y.J. Borchers C. Xiong Y. Nat. Cell Biol. 2003; 5: 1001-1007Crossref PubMed Scopus (332) Google Scholar). To determine whether BTB domain itself is sufficient for interaction with Cul3, various deletion mutants of SPOP were constructed (Fig. 1B, upper panel). HA-Cul3 was expressed in 293T cells with each of the FLAG-SPOP deletion mutants. The SPOP mutants that lack either the N-terminal MATH domain (indicated by m3) or the C-terminal region (m2) were co-immunoprecipitated with Cul3 (Fig. 1B, lower panel). Surprisingly, neither MATH domain (m1) nor BTB domain alone (m4) was capable of interacting with Cul3. Nor could the C-terminal region alone (m5) interact with Cul3. These results suggest that the N- or C-terminal sequence adjacent to BTB domain may also contribute to the interaction of SPOP with Cul3. Fig. 1C confirms that SPOP specifically interacts with Cul3 but not with the other Cul family members. Notably, SPOP ran as a doublet on the gel when expressed with Cul3 but not with the other Culs, and only one of the two bands interacted with Cul3. Therefore, we examined which of the SPOP doublet interacts with Cul3. We also examined whether SPOP interacts with any or both of Cul3 and its Nedd8-conjugated form and whether Daxx interacts with any or both of the SPOP doublet. Immunoprecipitation analysis shows that Cul3 interacted only with the slow-migrating form of SPOP (Fig. 1D, left panels), SPOP interacts with both Cul3 and its Nedd8-conjugated form (middle panel), and Daxx interacts with both of the SPOP doublet (right panel). However, the nature of the fast-migrating form of SPOP remains unknown, although it is unlikely that the fast-migrating form is a cleavage product of SPOP whose C-terminal fragment was removed, since the SPOP mutant (m2) that lacks the C-terminal 297-375 sequence is still capable of interacting with Cul3.SPOP-mediated Interaction of Daxx with Cul3—Daxx was shown to interact with the N-terminal MATH domain of SPOP (35La M. Kim K. Park J. Won J. Lee J.H. Fu Y.M. Meadows G.G. Joe C.O. Biochem. Biophys. Res. Commun. 2004; 320: 760-765Crossref PubMed Scopus (16) Google Scholar). To determine whether SPOP can act as an adaptor for the interaction between Cul3 and Daxx, FLAG-Cul3 was expressed in 293T cells with HA-Daxx or both FLAG-SPOP and HA-Daxx. Immunoprecipitation with anti-HA antibody shows that Daxx interacts with Cul3 in the presence of SPOP but much more weakly in its absence (Fig. 2A). To confirm the requirement of SPOP for the interaction between Daxx and Cul3, HA-Daxx was expressed with FLAG-Cul3 or both HA-SPOP and FLAG-Cul3. Immunoprecipitation with anti-FLAG antibody again shows that Cul3 interacts with Daxx in the presence of SPOP but much more weakly in its absence (Fig. 2B). We then examined whether endogenous SPOP is also capable of interacting with Cul3 and Daxx. Lysates were obtained from 293T cells that had been treated with MG132. Immunoprecipitation of cell lysates with anti-SPOP antibody shows that SPOP interacts with both endogenous Cul3 and Daxx (Fig. 2C). Thus, it is likely that the weak interaction between ectopically expressed Cul3 and Daxx, observed without overexpression of SPOP, is mediated by endogenous SPOP. To clarify further whether the interaction of Cul3 with Daxx is mediated by SPOP but not by their direct binding, HA-Daxx, MycHis-SPOP, and FLAG-Cul3 were in vitro translated in the presence of [35S]Met. The labeled proteins were then subjected to immunoprecipitation with anti-HA antibody. Cul3 was co-precipitated with Daxx only when SPOP is present (Fig. 2D), suggesting that Cul3 does not directly bind to Daxx. Collectively, these results demonstrate that SPOP functions as an adaptor protein that bridges Daxx to Cul3.FIGURE 2SPOP-mediated interaction of Daxx with Cul3. A, FLAG-Cul3 was expressed in 293T cells with HA-Daxx or both FLAG-SPOP and HA-Daxx. After incubation for 24 h, cells were treated with 20 μm MG132 for the next 4 h. Cell lysates were then subjected to immunoprecipitation (IP) with anti-HA antibody followed by immunoblot with anti-HA or anti-FLAG antibody. The lysates were also probed with respective antibodies. B, HA-Daxx was expressed in cells with FLAG-Cul3 or both HA-SPOP and FLAG-Cul3. After culturing the cells as above, cell lysates were subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblot with anti-HA or anti-FLAG antibody. The lysates were also probed with respective antibodies. C, 293T cells were treated with 20 μm MG132 for 4 h. Cell lysates were prepared and subjected to immunoprecipitation with anti-SPOP antibody followed by immunoblot with anti-Daxx, anti-Cul3, or anti-SPOP antibody. D, indicated combinations of in vitro translated 35S-labeled HA-Daxx, MycHis-SPOP, and FLAG-Cul3 were incubated for 2 h at 4°C. After incubation, the samples were subjected to immunoprecipitation with anti-HA antibody followed by autoradiography. The asterisks indicate a nonspecific band.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether Daxx co-localizes in cells with Cul3 through their interaction with SPOP, HeLa cells were transfected with each or varied combinations of expression vectors for the proteins. Immunocytochemical analysis reveals that Cul3, which alone localizes predominantly in the cytoplasm (Fig. 3A), was recruited to the nucleus and co-localized as speckles with SPOP upon co-expression with SPOP (Fig. 3B). Likewise, Daxx, which alone resides throughout the nucleoplasm, was recruited to nuclear speckles upon co-expression with SPOP. Co-expression of Daxx with both Cul3 and SPOP resulted in co-localization of all three proteins within the nucleus but as clump-like structures that appear distinct from nuclear speckles. Without expression of SPOP, however, Cul3 remained in the cytoplasm, whereas Daxx was found throughout the nucleoplasm as if each was expressed separately. These results further demonstrate that SPOP serves as an adaptor molecule that recruits Cul3 to the nucleus for interaction with Daxx.FIGURE 3Co-localization of Cul3, Daxx, and SPOP. Each (A) or indicated combinations of differentially tagged Daxx, Cul3, and SPOP (B) were expressed in HeLa cells. Cells were then stained with respective antibodies. The bar indicates 10 μm. DAPI, 4,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; YFP, yellow fluorescent protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SPOP/Cul3-mediated Degradation of Daxx—Although it was unknown whether Daxx is degraded in cells, the level of Daxx protein has been shown to decrease when cells are induced for apoptosis (28Chen L.Y. Chen J.D. Mol. Cell. Biol. 2003; 23: 7108-7121Crossref PubMed Scopus (97) Google Scholar). In an attempt to determine whether Daxx is ubiquitinated by Cul3-based Ub ligase for degradation by the proteasome, Cul3 or its mutant forms were expressed in 293T cells with SPOP and Roc1. Fig. 4A shows that Daxx level is markedly reduced by expression of wild-type Cul3 but much less significantly by that of Y62G or K711S. These results suggest that the degradation of Daxx requires the interaction of Cul3 with SPOP in addition to Nedd8 modification of Cul3. However, Daxx level in cells expressing Y62G or K711S with SPOP was also lower than that in cells expressing Daxx only. Thus, the partial degradation of Daxx that occurred in cells expressing Y62G or K711S with SPOP might be mediated by the interaction of endogenous Cul3 with SPOP.FIGURE 4Requirement of SPOP for Daxx degradation. A, FLAG-tagged wild-type Cul3, Y62G, or K711S was expressed in 293T cells with HA-Daxx, FLAG-SPOP, and FLAG-Roc1. Cell lysates were subjected to immunoblot with respective antibodies. As a control, β-actin was identified by using anti-β-actin antibody. B, FLAG-tagged SPOP (wt) or its deletion mutants (m2 or m3) were expressed in cells with HA-Daxx, FLAG-Cul3, and FLAG-Roc1. They were then probed as above. C, HA-Daxx was expressed in cells with or without FLAG-tagged SPOP, Cul3, an

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