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• W2018006434 abstract "The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes. The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes. The von Hippel-Lindau (VHL) 1The abbreviations used are: VHL, von Hippel-Lindau; VBC, the VHL-Elongin BC complex; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; SCF, Skp1-Cul1/Cdc53-F-box; Ub, ubiquitin; h, human; E1, Ub-activating enzyme; HIF, hypoxia-inducible factor; HA, hemagglutinin; PDB, Protein Data Bank. tumor suppressor complex is the founding member of the family of Elongin BC-containing E3 ubiquitin ligases, which are composed of a substrate recognition subunit, Elongins B and C, a member of the Cullin family of proteins (either Cul2 or Cul5), and the RING finger protein Rbx1 (also known as ROC1 or Hrt1) (1Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 2Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner R.D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441Crossref PubMed Scopus (429) Google Scholar, 3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar, 4Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (667) Google Scholar). The VHL protein is one of a large family of proteins that bind Elongins B and C through a conserved BC box motif, which is a 10-amino-acid degenerate motif of sequence (A,P,S,T)LXXXCXXX(A,-I,L,V) (5Kamura T. Sato S. Haque D. Liu L. Kaelin W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (503) Google Scholar, 6Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (527) Google Scholar, 7Kamura T. Burian D. Yan Q. Schmidt S.L. Lane W.S. Querido E. Branton P.E. Shilatifard A. Conaway R.C. Conaway J.W. J. Biol. Chem. 2001; 276: 29748-29753Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 8Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (574) Google Scholar, 9Aso T. Haque D. Barstead R.J. Conaway R.C. Conaway J.W. EMBO J. 1996; 15: 5557-5566Crossref PubMed Scopus (90) Google Scholar). In the context of the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit and is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that functions as a potent activator of ubiquitylation of target proteins by an E2 ubiquitin-conjugating enzyme. Elongin B, a ubiquitin-like protein, associates with the complex through interactions with Elongin C and appears to stabilize the binding of Elongin C to VHL. The best characterized substrates of the VHL ubiquitin ligase are the HIFα family of DNA binding transcription factors (1Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar, 2Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner R.D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441Crossref PubMed Scopus (429) Google Scholar, 3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar, 10Maxwell P.H. Wiggener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4152) Google Scholar, 11Lisztwan J. Imbert G. Wirbelauer C. Gstaiger M. Krek W. Genes Dev. 1999; 13: 1822-1833Crossref PubMed Scopus (340) Google Scholar), which function together with the constitutively expressed aryl hydrocarbon nuclear translocator to regulate expression of hypoxia-inducible genes when cells are starved for oxygen (12Semenza G.L. Annu. Rev. Cell Dev. Biol. 2000; 15: 551-578Crossref Scopus (1676) Google Scholar). Cellular HIFα protein levels are tightly controlled by the ubiquitin-dependent proteolytic system (13Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1849) Google Scholar, 14Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). In cells grown in a plentiful supply of oxygen, HIFα family members are hydroxylated at critical proline residues within their oxygen-dependent degradation domains (15Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3893) Google Scholar, 16Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Griegsheim A.V. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4456) Google Scholar, 17Yu F. White S.B. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (647) Google Scholar, 18Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2114) Google Scholar, 19Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2736) Google Scholar, 20Ivan M. Haberberger T. Gervasi D.C. Michelson K.S. Gunzler V. Kondo K. Yang H. Sorokina I. Conaway R.C. Conaway J.W. Kaelin W.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13459-13464Crossref PubMed Scopus (491) Google Scholar). Upon VHL binding to their hydroxylated oxygen-dependent degradation domains, HIFα family members are rapidly ubiquitylated by the VHL ubiquitin ligase complex and degraded by the proteasome. In cells grown in hypoxic conditions, HIFα family members are not hydroxylated. Accordingly, under hypoxic conditions, the VHL ubiquitin ligase complex cannot bind HIFαs and promote their ubiquitylation and degradation, so cellular levels of these transcription factors rise, leading to activation of hypoxically regulated genes. The large family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases share striking structural similarities with the VHL ubiquitin ligase. SCF ubiquitin ligases are composed of one of many F-box proteins, the Elongin C-like protein Skp1, Cullin family member Cul1 (called Cdc53 in the Saccharomyces cerevisiae), and Rbx1 (4Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (667) Google Scholar, 21Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (984) Google Scholar, 22Feldman R.M. Correll C.C. Kaplan K.B. Deshaies R.J. Cell. 1997; 91: 221-230Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, 23Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. 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Deshaies R.J. Genes Dev. 1999; 13: 1614-1626Crossref PubMed Scopus (355) Google Scholar, 30Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1083) Google Scholar). In the context of SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits and are linked by the adaptor protein Skp1 to a Cul1/Rbx1 module that activates an E2 to ubiquitylate target proteins. Many substrates of SCF ubiquitin ligases have been characterized, and ubiquitylation of most, but not all, depends upon the E2 ubiquitin-conjugating enzyme Cdc34 (30Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1083) Google Scholar, 31Pickart C. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar, 32Yaron A. Hatzubai A. Davis M. Lavon I. Amit S. Manning A.M. Andersen J.S. Mann M. Mercurio F. Ben-Neriah Y. Nature. 1998; 396: 590-594Crossref PubMed Scopus (571) Google Scholar, 33Oh K.J. Kalinina A. Wang J. Nakayama K. Nakayama K.I. Bagchi S. J. Virol. 2004; 78: 5338-5346Crossref PubMed Scopus (69) Google Scholar, 34Strack P. Caligiuri M. Pelletier M. Boisclair M. Theodoras A. Beer-Romero P. Glass S. Parsons T. Copeland R.A. Auger K.R. Benfield P. Brizuela L. Rolfe M. Oncogene. 2000; 19: 3529-3536Crossref PubMed Scopus (35) Google Scholar). In contrast, at least in vitro, VHL ubiquitin ligase-dependent ubiquitylation of HIFαs depends on members of the Ubc5 family of E2s (2Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner R.D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441Crossref PubMed Scopus (429) Google Scholar, 3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar). Our laboratory is currently engaged in biochemical studies to define the functions of subunits of VHL and SCF ubiquitin ligases. In this report, we investigate the functions of the Elongin C and Skp1 adaptor proteins in reconstitution of active VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, by exploiting altered specificity Elongin C-Skp1 and Cdc4-VHL chimeric proteins, we provide insights into the determinants of E2 selectivity of VHL and SCF ubiquitin ligases. Antibodies—Anti-VHL monoclonal antibody (Ig32) was purchased from BD PharMingen. Anti-Myc (9E10) and anti-HA (12CA5) monoclonal antibodies were obtained from Roche Applied Science. Anti-HSV monoclonal antibody was from Novagen. Anti-FLAG monoclonal antibody (M2) was purchased from Sigma. Anti-Cul2 and anti-Elongin C monoclonal antibodies were obtained from BD Transduction Laboratories. Anti-Cul2 (CT2) rabbit polyclonal antibodies were from Zymed Laboratories Inc.. Anti-HIF1α monoclonal antibody (H1α67) was purchased from Novus Biologicals. Anti-protein C monoclonal antibody (HPC4) (35Stearns D.J. Kurosawa S. Sims P.J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1988; 263: 826-832Abstract Full Text PDF PubMed Google Scholar) was provided by C. Esmon (Oklahoma Medical Research Foundation) and is commercially available as anti-protein C from Roche Applied Science. Anti-Elongin B polyclonal antibody was prepared as described (36Garrett K.P. Aso T. Bradsher J.N. Foundling S.I. Lane W.S. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7172-7176Crossref PubMed Scopus (91) Google Scholar). Expression of Recombinant Proteins in Sf21 Insect Cells—Wild type human Elongin C and Elongin C mutants containing N-terminal epitope tags recognized by the HPC4 monoclonal antibody were subcloned into pBacPAK8. Recombinant baculoviruses were generated with the BacPAK baculovirus expression system (Clontech). Baculoviruses encoding human Cul1 and Cul2 containing N-terminal HA epitope tags were described previously (7Kamura T. Burian D. Yan Q. Schmidt S.L. Lane W.S. Querido E. Branton P.E. Shilatifard A. Conaway R.C. Conaway J.W. J. Biol. Chem. 2001; 276: 29748-29753Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Baculoviruses encoding human Cul2, human VHL, human VHL containing an N-terminal His6 tag, human Elongin B and mouse Rbx1 containing N-terminal Myc epitope tags, and human HIF1α containing N-terminal His6 and HPC4 tags were described previously (3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar). Baculoviruses encoding S. cerevisiae Cdc53 and mouse Rbx1 containing N-terminal His6 and Myc tags were described previously (4Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (667) Google Scholar). Sf21 cells were cultured at 27 °C in Sf-900 II SFM with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). Plates containing 1 × 107 Sf21 cells were infected with the recombinant baculoviruses indicated in the text and figures. Sixty hours after infections, cells were lysed in 1 ml/plate of ice-cold buffer containing 40 mm Hepes-NaOH (pH 7.9), 150 mm NaCl, 1 mm dithiothreitol, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol, 5 μg/ml leupeptin, 5 μg/ml antipain, 5 μg/ml pepstatin A, and 5 μg/ml aprotinin. Immunoprecipitations and Western Blotting—Approximately 100 μg of Sf21 cell lysates was incubated at 4 °C for 2 h with either 10 μl of protein G-Sepharose and 2 μg of HPC4 antibody or 10 μl of protein A-Sepharose and 2 μg of the antibodies indicated in the figures. Sepharose beads were washed three times in buffer containing 40 mm Hepes-NaOH (pH 7.9), 150 mm NaCl, 1 mm dithiothreitol, and 0.5% (v/v) Triton X-100. Immunoprecipitated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Hybond P membranes (Amersham Biosciences). Membranes were incubated at 4 °C overnight with the indicated antibodies in buffer containing 40 mm Tris-HCl (pH 7.6), 100 mm NaCl, and 3% (w/v) nonfat dry milk and then with peroxidase-conjugated secondary antibodies (Sigma). Proteins were visualized on film following treatment of membranes with Supersignal West Pico, Dura, or Femto chemiluminescence reagent (Pierce). In some experiments, proteins were visualized with a Storm gel and blot imaging system following treatment of membranes with ECL plus Western blotting reagents (Amersham Biosciences). Alternatively, membranes were incubated at 4 °C overnight with the indicated antibodies in Odyssey blocking solution (LI-COR Biosciences) and then with the appropriate Alexa Fluor 680 (Molecular Probes) or IRDye 800 (Rockland) secondary antibodies. In this case, proteins were visualized and quantitated with the Odyssey infrared imaging system (LI-COR Biosciences). Purification of Recombinant VHL and Cullin/Rbx1 Complexes from Sf21 Cell Lysates—Plates containing 1 × 107 Sf21 cells were coinfected with the recombinant baculoviruses indicated in the text and figures. Sixty hours after infections, cells were collected by centrifugation and resuspended in 1 ml/plate of ice-cold buffer containing 40 mm Hepes-NaOH (pH 7.9), 150 mm NaCl, 5 μg/ml leupeptin, 5 μg/ml antipain, 5 μg/ml pepstatin A, 5 μg/ml aprotinin, and 40 mm imidazole (pH 7.9). Cells were lysed by French press (1-inch piston; 16,000 p.s.i. of pressure; American Instrument Company). Following centrifugation at 10,000 × g for 20 min at 4 °C, the resulting supernatant was mixed with 1 ml of Ni+2-agarose pre-equilibrated in buffer containing 40 mm Hepes-NaOH (pH 7.9), 150 mm NaCl, and 40 mm imidazole (pH 7.9). The slurry was incubated at 4 °C for 2 h with gentle mixing, washed three times with the same buffer, and packed into a 0.8 cm-diameter column. The column was eluted stepwise with buffer containing 40 mm Hepes-NaOH (pH 7.9), 50 mm NaCl, 10% (v/v) glycerol, and 300 mm imidazole (pH 7.9). VBC-Cullin/Rbx1 complexes were reconstituted by mixing Ni+2-agarose-purified VHL-Elongin BC complex (VBC) and Cullin/Rbx1 together and incubating them for 1 h at 4 °C. For the experiment in Fig. 5 (see below), VBC-Cul2/Rbx1 complex was further purified by TSK DEAE-NPR HPLC. Ni+2-agarose fractions were dialyzed against buffer containing 40 mm Tris-HCl (pH 7.9), 100 mm KCl, 1 mm dithiothreitol, 0.5 mm EDTA, and 10% (v/v) glycerol. Following centrifugation at 10,000 × g for 20 min at 4 °C, the resulting supernatant was applied to a TSK DEAE-NPR HPLC column (4.6 × 35 mm; Toso-Haas) preequilibrated in the same buffer. The column was eluted at 0.2 ml/min with a 3-ml linear gradient from 100 to 500 mm KCl in the same buffer, and 0.1-ml fractions were collected. For the experiment of Fig. 7 (see below), complexes containing Cdc4-VHL, Elongins B and C, and Cul2/Rbx1 were purified by the same procedure.Fig. 7Ubiquitylation of phosphorylated Sic1 by an Elongin BC- and Cul2-containing ubiquitin ligase by hUbc5a.A, the Cdc4-VHL chimera contains residues 341–779 of Cdc4 fused to the complete VHL open reading frame. The Cdc4 F-box is indicated by the black box, the Cdc4 WD repeats are indicated by the dark gray boxes, and the VHL BC box is indicated by the white box. WT, wild type. B, ubiquitin ligase complexes containing either Cdc4 or Cdc4-VHL as F-box protein were expressed in insect cells and purified as described. ElobB, Elongin B; ElobC, Elongin C. C, purified Cdc4-VHL-BC-Cul2/Rbx1 complexes were assayed for their abilities to support ubiquitylation of phosphorylated HPC4-Sic1 in the presence of ∼100 ng of hUbc5a or yCdc34. Reaction products were analyzed by Western blotting (WB) with HPC4 monoclonal antibody and visualized on film. D, purified Cdc4-VHL-BC-Cul2/Rbx1 or VBC-Cul2/Rbx1 complexes were assayed as in panel C for their abilities to support ubiquitylation of phosphorylated HPC4-Sic1.View Large Image Figure ViewerDownload (PPT) Preparation of Hydroxylated HIF1α—Sf21 cells were infected with baculoviruses encoding human HIF1α containing N-terminal His6 and HPC4 tags. HIF1α was purified from Sf21 cell lysates by Ni+2-agarose chromatography as described previously (3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar). Approximately 5 μg of purified HIF1α was incubated at room temperature for 2 h with 200 μl of TnT wheat germ extract translation system (Promega) programmed with a pcDNA3 expression vector encoding the EGLN1 prolyl hydroxylase (20Ivan M. Haberberger T. Gervasi D.C. Michelson K.S. Gunzler V. Kondo K. Yang H. Sorokina I. Conaway R.C. Conaway J.W. Kaelin W.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13459-13464Crossref PubMed Scopus (491) Google Scholar) and containing 100 μm FeCl2, 2 mm sodium ascorbate, and 5 mm α-ketoglutarate. Prolyl hydroxylated HIF1α was then purified from the reaction mixture by Ni+2-agarose chromatography (3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar). Assays of HIF1α and Sic1 Ubiquitylation in Vitro—Human Ubc5a (hUbc5a), human Cdc34 (hCdc34), and S. cerevisiae Cdc34 (yCdc34), all containing N-terminal His6 tags, S. cerevisiae Uba1 (yUba1) containing N-terminal Myc and His6 tags, and mammalian GST·ubiquitin were prepared as described (3Kamura T. Sato S. Iwai K. Czyzyk-Krezeska M.F. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (551) Google Scholar). Reconstituted VBC-Cullin/Rbx1 complexes or aliquots of TSK DEAE-NPR HPLC-purified VBC-Cul2/Rbx1 or VHL-Cdc4BC-Cul2/Rbx1 complexes were incubated with 50 ng of yUba1, 500 ng of GST·Ub, 20 ng of purified prolyl hydroxylated HIF1α or phosphorylated Sic1 (23Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. Cell. 1997; 91: 209-219Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar), and varying amounts of the E2 ubiquitin-conjugating enzymes hUbc5a, hUbc3, or yCdc34 as indicated in the text and figures in 10 μl of ubiquitylation reaction buffer containing 40 mm Hepes-NaOH (pH 7.9), 60 mm potassium acetate, 2 mm dithiothreitol, 6 mm MgCl2, 0.5 mm EDTA (pH 8.0), 10% (v/v) glycerol, and 1.5 mm ATP. Reaction mixtures were incubated for 1 h at 25 °C, fractionated by SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with anti-HIF1α antibodies. Assay of E2-Ubiquitin Thioester Bond Formation—hUbc5a, hUbc3, or yCdc34 was incubated with 50 ng of yUba1 and 100 ng of His-T7-Xpress-ubiquitin in 10 μl of 40 mm Hepes-NaOH (pH 7.9), 60 mm potassium acetate, 2 mm dithiothreitol, 6 mm MgCl2, 0.5 mm EDTA (pH 8.0), 10% (v/v) glycerol, and 1.5 mm ATP. Reaction mixtures were incubated for 1 h at 25 °C and then stopped with 15 μl of a buffer containing 60 mm Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and either 30 mm dithiothreitol (reducing) or 6 m urea (nonreducing). Samples were incubated at 100 °C for 3 min (reducing) or 37 °C for 15 min (nonreducing), subjected to SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with anti-T7 antibodies. Computational Methods—Multiple sequence alignments were made using the MACAW program (37Schuler G.D. Altschul S.F. Lipman D.J. Proteins Struct. Funct. Genet. 1991; 9: 180-190Crossref PubMed Scopus (895) Google Scholar). Homology modeling and loop rebuilding were done on the Swiss-Model server (38Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4506) Google Scholar). Automated docking was done using the GRAMM program (39Katchalski-Katzir E. Shariv I. Eisenstein M. Friesem A.A. Alflalo C. Vakser I.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (863) Google Scholar). Fig. 8 (see below) was generated with the PyMOL package (DeLano Scientific, LLC; www.pymol.org). As illustrated in Fig. 1, the Elongin C and Skp1 proteins share significant amino acid sequence similarity and belong to a protein superfamily with shared three-dimensional structure. Elongin C and Skp1 function as adaptors that recruit heterodimeric Cul2/Rbx1 and Cul1/Rbx1 modules, respectively, into VHL and SCF ubiquitin ligase complexes. In the case of the VHL ubiquitin ligase, Elongin C recruits a Cul2/Rbx1 module to the complex by interacting specifically with a BC box in the VHL protein (Ref. 40Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (693) Google Scholar and references therein) and with an N-terminal Cul2 region (41Pause A. Peterson B. Schaffar G. Stearman R. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9533-9538Crossref PubMed Scopus (81) Google Scholar). In the case of SCF ubiquitin ligase complexes, Skp1 functions by an apparently similar mechanism as Elongin C to recruit a Cul1/Rbx1 module to the complex by interacting specifically with an F-box in F-box proteins and with an N-terminal Cul1 region (42Wu K. Fuchs S.Y. Chen A. Tan P. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. Biol. 2000; 20: 1382-1393Crossref PubMed Scopus (92) Google Scholar, 43Zheng 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 (1170) Google Scholar). To begin to study the relationship between the roles of Elongin C and Skp1 in reconstitution of their respective ubiquitin ligases, we sought to identify Elongin C sequences required for its interaction with Cul2 and for its recruitment of the Cul2/Rbx1 module into the VHL complex. To accomplish this, we constructed baculoviruses encoding a systematic series of Elongin C deletion mutants and analyzed the abilities of these Elongin C mutants to support the assembly of VBC and of the complete VBC-Cul2/Rbx1 ubiquitin ligase in Sf21 insect cells. Sf21 cells were coinfected with baculoviruses encoding VHL, Elongin B, Cul2, Rbx1, and wild type or mutant Elongin C with an N-terminal epitope tag recognized by the HPC4 monoclonal antibody. Elongin C-containing complexes were immunoprecipitated with HPC4 monoclonal antibodies and analyzed by Western blotting for the presence of individual subunits of the VHL complex. As shown in Fig. 2A, recruitment of Cul2 to the VHL complex was strongly dependent on both Elongins B and C. Elongin C was quite sensitive to deletions. Only three of the mutants could assemble similarly to wild type Elongin C into the VBC complex; two of these, Δ41–50 and Δ51–60, were defective in recruitment of Cul2 into the VHL ubiquitin ligase complex (Fig. 2B). These results suggested that Elongin C sequences between residues 41 and 60 are not crucial for VBC assembly but are important for Elongin C interaction with Cul2 and recruitment of Cul2 into the VHL ubiquitin ligase complex. Notably, some residues within the corresponding region of Skp1 appear to contribute to formation of the Skp1-Cul1 interface (43Zheng 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 (1170) Google Scholar). To investigate further the role of Elongin C sequences 41–60 in recruitment of Cul2 into the VHL ubiquitin ligase, we carried out domain swaps between Elongin C and Skp1 in an effort to identify an Elongin C-Skp1 chimeric protein capable of recruiting a Cul1/Rbx1 module into the VHL complex. In one mutant, designated M2, Elongin C residues 40–47 were replaced with Skp1 residues 25–32. These stretches of sequence in Elongin C and Skp1 fall largely within conserved helix 2 (Fig. 1) (40Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (693) Google Scholar, 44Schulman B.A. Carrano A.c. Jeffrey P.D. Bowen Z. Kinnucan E.R.E. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. Nature. 2000; 408: 381-386Crossref PubMed Scopus (490) Google Scholar). A second Elongin C-Skp1 chimeric protein, designated M1, was created by replacing Elongin C residues 47–57 with Skp1 residues 32–42. Elongin C residues 47–57 form a loop (loop 3) between helix 2 and strand 3 in human Elongin C; loop 3 was poorly resolved in the Elongin C x-ray structures (40Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (693)" @default.
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