Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase

Science

Volumn 284, Issue 5414, 1999, Pages 657-661

  Kamura, T ^a   Koepp, D M ^a   Conrad, M N ^a   Skowyra, D ^a   Moreland, R J ^a   Iliopoulos, O ^a   Lane, W S ^a   Kaelin Jr , W G ^a   Elledge, S J ^a   Conaway, R C ^a   Harper, J W ^a   Conaway, J W ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CELL PROTEIN; PROTEIN RBX1; REPRESSOR PROTEIN; UBIQUITIN PROTEIN LIGASE; UNCLASSIFIED DRUG; VON HIPPEL LINDAU PROTEIN;

ANIMAL TISSUE; ARTICLE; CELL CYCLE G1 PHASE; CELL CYCLE S PHASE; GENE MUTATION; KIDNEY CANCER; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROTEIN PROCESSING; PROTEIN PROTEIN INTERACTION; RAT; SEQUENCE HOMOLOGY; TRANSCRIPTION REGULATION; TUMOR SUPPRESSOR GENE; VON HIPPEL LINDAU DISEASE;

AMINO ACID SEQUENCE; ANIMALS; CARRIER PROTEINS; CELL CYCLE; CELL CYCLE PROTEINS; CELL LINE; CULLIN PROTEINS; F-BOX PROTEINS; FUNGAL PROTEINS; LIGASES; LIVER; MALE; MOLECULAR SEQUENCE DATA; PEPTIDE SYNTHASES; PROTEINS; RATS; RATS, SPRAGUE-DAWLEY; RECOMBINANT FUSION PROTEINS; S-PHASE KINASE-ASSOCIATED PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SEQUENCE ALIGNMENT; SKP CULLIN F-BOX PROTEIN LIGASES; TRANSCRIPTION FACTORS; TUMOR SUPPRESSOR PROTEINS; UBIQUITIN-PROTEIN LIGASES; UBIQUITINS; VON HIPPEL-LINDAU TUMOR SUPPRESSOR PROTEIN;

ANIMALIA;

EID: 0033597443     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5414.657     Document Type: Article

References (41)

1. W. M. Linehan, M. I. Lerman, B. Zbar, J. Am. Med. Assoc. 273, 564 (1995); J. R. Gnarra et al., Biochim. Biophys. Acta 1242, 201 (1996); W. G. Kaelin and E. R. Maher, Trends. Genet. 14, 423 (1998).
2. W. M. Linehan, M. I. Lerman, B. Zbar, J. Am. Med. Assoc. 273, 564 (1995); J. R. Gnarra et al., Biochim. Biophys. Acta 1242, 201 (1996); W. G. Kaelin and E. R. Maher, Trends. Genet. 14, 423 (1998).
3. W. M. Linehan, M. I. Lerman, B. Zbar, J. Am. Med. Assoc. 273, 564 (1995); J. R. Gnarra et al., Biochim. Biophys. Acta 1242, 201 (1996); W. G. Kaelin and E. R. Maher, Trends. Genet. 14, 423 (1998).
4. O. Iliopoulos, A. P. Levy, C. Jiang, W. G. Kaelin, M. A. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 93, 10595 (1996); J. R. Gnarra et al., ibid., p. 10589; G. Siemeister et al., Cancer Res. 56, 2299 (1996).
5. O. Iliopoulos, A. P. Levy, C. Jiang, W. G. Kaelin, M. A. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 93, 10595 (1996); J. R. Gnarra et al., ibid., p. 10589; G. Siemeister et al., Cancer Res. 56, 2299 (1996).
6. O. Iliopoulos, A. P. Levy, C. Jiang, W. G. Kaelin, M. A. Goldberg, Proc. Natl. Acad. Sci. U.S.A. 93, 10595 (1996); J. R. Gnarra et al., ibid., p. 10589; G. Siemeister et al., Cancer Res. 56, 2299 (1996).
7. A. Pause, S. Lee, K. M. Lonergan, R. D. Klausner, Proc. Natl. Acad. Sci. U.S.A. 95, 993 (1998); M. Kim et al., Biochim. Biophys. Res. Commun. 253, 672 (1999).
8. A. Pause, S. Lee, K. M. Lonergan, R. D. Klausner, Proc. Natl. Acad. Sci. U.S.A. 95, 993 (1998); M. Kim et al., Biochim. Biophys. Res. Commun. 253, 672 (1999).
9. M. Ohh et al., Mol. Cell 1, 959 (1998).
10. D. R. Duan et al., Science. 269, 1402 (1995); A. Kibel, O. Iliopoulos, J. A. DeCaprio, W. G. Kaelin Jr., ibid., p. 1444.
11. D. R. Duan et al., Science. 269, 1402 (1995); A. Kibel, O. Iliopoulos, J. A. DeCaprio, W. G. Kaelin Jr., ibid., p. 1444.
12. A. Pause et al., Proc. Natl. Acad. Sci. U.S.A. 94, 2156 (1997); K. M. Lonergan et al., Mol. Cell. Biol. 18, 732 (1998).
13. A. Pause et al., Proc. Natl. Acad. Sci. U.S.A. 94, 2156 (1997); K. M. Lonergan et al., Mol. Cell. Biol. 18, 732 (1998).
14. The VHL complex was purified from a postnuclear supernatant prepared from the livers of 360 male Sprague-Dawley rats (∼3 kg of liver). Details of the purification are available at www.sciencemag.org/feature/data/991128.shl.
15. The VHL complex was fractionated by 13% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were visualized by staining the gel with Coomassie blue, excised, and subjected to in-gel reduction, S-carboxyamidomethylation, and tryptic digestion. Using 10% of the digestion mixture, we determined peptide sequences in a single run by microcapillary reversed-phase chromatography coupled to the electrospray Ionization source of a quadruple ion trap mass spectrometer (Finnigan LCQ, San Jose, CA). The Ion trap's online data-dependent scans allowed the automatic acquisition of high-resolution spectra to determine charge state and exact mass, and tandem mass spectrometry spectra for sequence information. The relative collision energy was 35% and isolation width was 2.5 dalton. Identification of human and mouse expressed sequence tags that encoded the peptide sequences NHIMDLCIECQAN, QVCPLD-NREWEFQK, WNAVAL, and WLK was facilitated with the algorithm SEQUEST [J. K. Eng, A. L. McCormick, J. R. Yates III, J. Am. Soc. Mass Spectrom. 5, 976 (1994)] and by programs developed in the Harvard Microchemistry Facility [H. S. Chittum et al., Biochemistry 37, 10866 (1998)]. IMAGE Consortium cDNA clones [G. Lennon, C. Auffray, M. Polymeropoulos, M. B. Soares, Genomics 33, 151 (1996)] encoding the complete 108-amino acid ORFs of human (H71993) and mouse (W66989 and AA260839) Rbx1 were obtained from Research Genetics, (Huntsville, AL), and the nucleotide sequences of both strands were determined. Human (AF140598) and mouse (AF140599) cDNAs encoded identical polypeptides of 108 amino acids.
16. The VHL complex was fractionated by 13% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were visualized by staining the gel with Coomassie blue, excised, and subjected to in-gel reduction, S-carboxyamidomethylation, and tryptic digestion. Using 10% of the digestion mixture, we determined peptide sequences in a single run by microcapillary reversed-phase chromatography coupled to the electrospray Ionization source of a quadruple ion trap mass spectrometer (Finnigan LCQ, San Jose, CA). The Ion trap's online data-dependent scans allowed the automatic acquisition of high-resolution spectra to determine charge state and exact mass, and tandem mass spectrometry spectra for sequence information. The relative collision energy was 35% and isolation width was 2.5 dalton. Identification of human and mouse expressed sequence tags that encoded the peptide sequences NHIMDLCIECQAN, QVCPLD-NREWEFQK, WNAVAL, and WLK was facilitated with the algorithm SEQUEST [J. K. Eng, A. L. McCormick, J. R. Yates III, J. Am. Soc. Mass Spectrom. 5, 976 (1994)] and by programs developed in the Harvard Microchemistry Facility [H. S. Chittum et al., Biochemistry 37, 10866 (1998)]. IMAGE Consortium cDNA clones [G. Lennon, C. Auffray, M. Polymeropoulos, M. B. Soares, Genomics 33, 151 (1996)] encoding the complete 108-amino acid ORFs of human (H71993) and mouse (W66989 and AA260839) Rbx1 were obtained from Research Genetics, (Huntsville, AL), and the nucleotide sequences of both strands were determined. Human (AF140598) and mouse (AF140599) cDNAs encoded identical polypeptides of 108 amino acids.
17. The VHL complex was fractionated by 13% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were visualized by staining the gel with Coomassie blue, excised, and subjected to in-gel reduction, S-carboxyamidomethylation, and tryptic digestion. Using 10% of the digestion mixture, we determined peptide sequences in a single run by microcapillary reversed-phase chromatography coupled to the electrospray Ionization source of a quadruple ion trap mass spectrometer (Finnigan LCQ, San Jose, CA). The Ion trap's online data-dependent scans allowed the automatic acquisition of high-resolution spectra to determine charge state and exact mass, and tandem mass spectrometry spectra for sequence information. The relative collision energy was 35% and isolation width was 2.5 dalton. Identification of human and mouse expressed sequence tags that encoded the peptide sequences NHIMDLCIECQAN, QVCPLD-NREWEFQK, WNAVAL, and WLK was facilitated with the algorithm SEQUEST [J. K. Eng, A. L. McCormick, J. R. Yates III, J. Am. Soc. Mass Spectrom. 5, 976 (1994)] and by programs developed in the Harvard Microchemistry Facility [H. S. Chittum et al., Biochemistry 37, 10866 (1998)]. IMAGE Consortium cDNA clones [G. Lennon, C. Auffray, M. Polymeropoulos, M. B. Soares, Genomics 33, 151 (1996)] encoding the complete 108-amino acid ORFs of human (H71993) and mouse (W66989 and AA260839) Rbx1 were obtained from Research Genetics, (Huntsville, AL), and the nucleotide sequences of both strands were determined. Human (AF140598) and mouse (AF140599) cDNAs encoded identical polypeptides of 108 amino acids.
18. W. Zachariae et al., Science 279, 1216 (1998).
19. 2-terminal His and HA tags, and recombinant baculoviruses were generated with the BacPAK baculovirus expression system (Clontech). The baculovirus vectors encoding 5. cerevisiae Cdc53 [A. R. Willems et al., Cell 86, 453 (1996)] and Elongins B and C have been described (29). Sf21 or Hi5 cells were cultured in Sf-900 II SFM with 5% fetal calf serum at 27°C and infected with the indicated recombinant baculoviruses. Sixty hours after infection, cells were collected and lysed in 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, and 5 μg/ml each of leupeptin, antipain, pepstatin A, and aprotinin. Lysates were centrifuged at 10,000g for 20 min at 4°C. The supernatants were used for immunoprecipitations.
20. Sources of antibodies were as follows: Anti-T7 and anti-HSV, Novagen; anti-HA (12CA5) and anti-c-MYC (9E10), Boehringer-Mannheim; anti-FLAG (M2), Eastman Kodak; anti-Elongin C monoclonal antibody (mAb), Transduction Laboratories; anti-VHL mAb (Ig32), Pharmingen; anti-Sic1 (yN-19 and yC-19) and anti-Cdc53 (yC-17), Santa Cruz Biotechnology. Anti-Elongin B rabbit polyclonal antibodies have been described previously (29). Anti-Sic1, Cdc4, and Skp1 were from (15). Immunoblotting and immunoprecipitations were performed as in (29).
21. 2-terminal 6-histidine and COOH-terminal FLAG epitope tags. Purification of recombinant proteins from Inclusion bodies and expression constructs for Elongins B and C have been described (29).
22. E. T. Kipreos, L. E. Lander, J. P. Wing, W. M. He, E. M. Hedgecock, Cell 85, 829 (1996).
23. C. Bai et al., ibid. 86, 263 (1996).
24. D. Skowyra, K. L. Craig, M. Tyers, S. J. Elledge, J. W. Harper, ibid. 91, 209 (1997).
25. R. M. R. Feldman, C. C. Cornell, K. B. Kaplan, R. J. Deshaies, ibid., p. 221.
26. E. E. Patton et al., Genes Dev. 12, 692 (1998).
27. E. E. Patton, A. R. Willems, M. Tyers, Trends Biochem. Sci. 14, 236 (1998).
28. E. Schwob, T. Boehm, M. D. Mendenhall, K. Nasmyth, Cell 79, 233 (1994).
29. A. Baudin, O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, C. Cullin, Nucleic Acids. Res. 21, 3329 (1993).
30. + transformants were selected. Random spores were germinated on galactose medium minus histidine and uracil and allowed to grow for 4 days at 30°C. The resulting colonies were tested for mating. To confirm that rescue was due to the presence of the RBX1 expression plasmid, cells were tested for the ability to grow in FOA (5-fluoro-orotic acid) after prolonged growth in medium containing uracil. The rbx1-1 ts allele was isolated as described (23). Strains carrying either a wild-type copy of RBX1 or rbx1-1 were transformed with either an empty GAL vector (pHY316) or construct expressing Sic1 under control of the GAL1, 10 promoter (pCB24) (14). Transformants were streaked to selective media containing either glucose or galactose at 30°C.
31. Less mRbx1 was immunoprecipitated from yeast containing the endogenous RBX1 gene (Fig. 3A, lane 3) than from yeast deleted for RBX1 (lane 1), even though similar amounts of Cdc53 were precipitated. mRbx1 was highly overexpressed in these cells and was likely present in large excess over the endogenous Cdc53.
32. D. Skowyra et al., Science 284, 662 (1999).
33. 6) infected with the indicated baculoviruses were lysed as described (15, 23) and immunoprecipitated with immobilized anti-MYC or anti-Flag. Complexes and lysates were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cdc34-dependent Sic1 ubiquitination assays were performed was described with Cln1/Cdc28-phosphorylated Sic1 as substrate (15). Sic1 ubiquitination assays in fractionated yeast lysates were performed essentially as described (31), except that FLAG-Skp1-Cdc4 complexes (200 ng) purified from insect cells with immobilized anti-FLAG were added to yeast lysates to stimulate Sic1 ubiquitination activity.
34. A. Yaron et al., Nature 396, 590 (1998).
35. F. Margottin et al., Mol. Cell 1, 565 (1998).
36. J. T. Winston et al., Genes Dev. 13, 270 (1999).
37. E. Spencer, J. Jiang, Z. J. Chen, ibid., p. 284.
38. T. Kamura et al., ibid. 12, 3872 (1998).
39. G. D. Schuler, S. F. Altschul, D. J. Lipman, Proteins Struct. Funct. Genet. 9, 180 (1991).
40. R. Verma, R. M. Feldman, R. J. Deshaies, Mol. Biol. Cell 8, 1427 (1997).
41. We thank M. Dresser for helpful discussions, K. Pierce and D. Kirby for mass spectrometry and sequencing, K. Jackson for oligonucleotide synthesis, and C. Esmon for anti-HPC4. Supported by NIH grants GM41628 (R.C.C.) and AG-11085 (J.W.H. and S.J.E.), the H. A. and Mary K. Chapman Charitable Trust (R.C.C. and J.W.C), the Welch Foundation (J.W.H.), and a Helen Hay Whitney Postdoctoral Fellowship (D.M.K). J.W.C., W.G.K., and S.J.E. are investigators of the Howard Hughes Medical Institute.