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• W2000789117 abstract "The product of the von Hippel-Lindau (VHL) tumor suppressor gene, pVHL, functions as a ubiquitin-protein isopeptide ligase in regulating HIF-1 protein turnover, thus accounting for the increased transcription of hypoxia-inducible genes that accompanies VHL mutations. The increased vascular endothelial growth factor mRNA stability in cells lacking pVHL has been hypothesized to be due to a similar regulation of an RNA-binding protein. We report the expression of the GLUT-1 3′-untranslated region RNA-binding protein, heteronuclear ribonucleoprotein (hnRNP) A2, is specifically increased in pVHL-deficient cell lines. Enhanced hnRNP A2 expression was apparent in all cell fractions, including polysomes, where a similar modest effect on hnRNP L (a GLUT-1 and VEGF 3′-untranslated region-binding protein), was seen. Steady state levels of hnRNP A2 mRNA were unaffected. Regulation of hnRNP A2 levels correlated with the ability of pVHL to bind elongin C. Proteasome inhibition of cells expressing wild type pVHL selectively increased cytoplasmic hnRNP A2 levels to that seen in pVHL-deficient cells. Finally, an in vivo interaction between pVHL and hnRNP A2 was demonstrated in both the nucleus and the cytoplasm. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL in a manner that is dependent on elongin C interactions as well as functioning proteasomes. The product of the von Hippel-Lindau (VHL) tumor suppressor gene, pVHL, functions as a ubiquitin-protein isopeptide ligase in regulating HIF-1 protein turnover, thus accounting for the increased transcription of hypoxia-inducible genes that accompanies VHL mutations. The increased vascular endothelial growth factor mRNA stability in cells lacking pVHL has been hypothesized to be due to a similar regulation of an RNA-binding protein. We report the expression of the GLUT-1 3′-untranslated region RNA-binding protein, heteronuclear ribonucleoprotein (hnRNP) A2, is specifically increased in pVHL-deficient cell lines. Enhanced hnRNP A2 expression was apparent in all cell fractions, including polysomes, where a similar modest effect on hnRNP L (a GLUT-1 and VEGF 3′-untranslated region-binding protein), was seen. Steady state levels of hnRNP A2 mRNA were unaffected. Regulation of hnRNP A2 levels correlated with the ability of pVHL to bind elongin C. Proteasome inhibition of cells expressing wild type pVHL selectively increased cytoplasmic hnRNP A2 levels to that seen in pVHL-deficient cells. Finally, an in vivo interaction between pVHL and hnRNP A2 was demonstrated in both the nucleus and the cytoplasm. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL in a manner that is dependent on elongin C interactions as well as functioning proteasomes. von Hippel-Lindau vascular endothelial growth factor glucose transporter 1 A+U-rich element heteronuclear ribonucleoprotein renal clear cell ubiquitin-protein isopeptide ligase hypoxia-inducible factor glyceraldehyde-3-phosphate dehydrogenase untranslated region 1,4-piperazinediethanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid horseradish peroxidase polyacrylamide gel electrophoresis Von Hippel-Lindau (VHL)1disease is an autosomal dominant cancer syndrome characterized by the predisposition to develop highly vascular tumors, including renal clear cell (RCC) carcinomas, cerebellar hemangioblastomas, retinal angiomata, and pheochromocytomas (1McKusick V.A. Mendelian Inheritance in Man. The Johns Hopkins University Press, Baltimore1992Google Scholar). Both germline as well as sporadic mutations of the VHL gene have been identified in patients afflicted with this disease (2Chen F. Kishida T. Yao M. Hustad T. Glavac D. Dean M. Gnarra J.R. Orcutt M.L. Duh F.M. Glenn G. Hum. Mutat. 1995; 5: 66-75Crossref PubMed Scopus (461) Google Scholar, 3Crossey P.A. Foster K. Richards F.M. Phipps M.E. Latif F. Tory K. Jones M.H. Bentley E. Kumar R. Lerman M.I. Hum. Genet. 1994; 93: 53-58Crossref PubMed Scopus (124) Google Scholar, 4Whaley J.M. Naglich J. Gelbert L. Hsia Y.E. Lamiell J.M. Green J.S. Collins D. Neumann H.P. Laidlaw J. Li F.P. Am. J. Hum. Genet. 1994; 55: 1092-1102PubMed Google Scholar, 5Tory K. Brauch H. Linehan M. Barba D. Oldfield E. Filling-Katz M. Seizinger B. Nakamura Y. White R. Marshall F.F. J. Natl. Cancer Inst. 1989; 81: 1097-1101Crossref PubMed Scopus (174) Google Scholar, 6Gnarra J.R. Tory K. Weng Y. Schmidt L. Wei M.H. Li H. Latif F. Liu S. Chen F. Duh F.M. Nat. Genet. 1994; 7: 85-90Crossref PubMed Scopus (1481) Google Scholar, 7Shuin T. Kondo K. Torigoe S. Kishida T. Kubota Y. Hosaka M. Nagashima Y. Kitamura H. Latif F. Zbar B. Cancer Res. 1994; 54: 2852-2855PubMed Google Scholar). Sporadic renal clear cell carcinomas are highly associated with mutation or transcriptional silencing of the VHL gene and subsequent loss or inactivation of the remaining VHL allele (6Gnarra J.R. Tory K. Weng Y. Schmidt L. Wei M.H. Li H. Latif F. Liu S. Chen F. Duh F.M. Nat. Genet. 1994; 7: 85-90Crossref PubMed Scopus (1481) Google Scholar, 7Shuin T. Kondo K. Torigoe S. Kishida T. Kubota Y. Hosaka M. Nagashima Y. Kitamura H. Latif F. Zbar B. Cancer Res. 1994; 54: 2852-2855PubMed Google Scholar). Thus, VHL conforms to Knudson's two-hit model of a tumor suppressor gene, in which gene inactivation occurs as the result of loss of function of both alleles (8Knudson Jr., A.G. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 820-823Crossref PubMed Scopus (5423) Google Scholar). The human VHL gene encodes a full-length protein of 213 amino acids which migrates with an apparent molecular mass of 30 kDa (9Latif F. Tory K. Gnarra J. Yao M. Duh F.M. Orcutt M.L. Stackhouse T. Kuzmin I. Modi W. Geil L. Science. 1993; 260: 1317-1320Crossref PubMed Scopus (2427) Google Scholar, 10Iliopoulos O. Kibel A. Gray S. Kaelin Jr., W.G. Nat. Med. 1995; 1: 822-826Crossref PubMed Scopus (590) Google Scholar). Internal translational initiation from an internal ATG at codon 54 produces a second 18-kDa gene product (11Iliopoulos O. Ohh M. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11661-11666Crossref PubMed Scopus (205) Google Scholar, 12Schoenfeld A. Davidowitz E. Burk R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8817-8822Crossref PubMed Scopus (186) Google Scholar, 13Blankenship C. Naglich J. Whaley J. Seizinger B. Kley N. Oncogene. 1999; 18: 1529-1535Crossref PubMed Scopus (103) Google Scholar). Both isoforms behave identically in all reports to date. VHL protein is predominantly expressed in the cytoplasmic compartments of most tissue and cell types, although it can shuttle between the nucleus and cytoplasm (10Iliopoulos O. Kibel A. Gray S. Kaelin Jr., W.G. Nat. Med. 1995; 1: 822-826Crossref PubMed Scopus (590) Google Scholar,14Duan D.R. Pause A. Burgess W. Aso T. Chen D.Y.T. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (506) Google Scholar, 15Lee S. Chen D.Y.T. Humphrey J.S. Gnarra J.R. Linehan W.M. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1770-1775Crossref PubMed Scopus (135) Google Scholar, 16Lee S. Neumann M. Stearman R. Stauber R. Pause A. Pavlakis G. Klausner R.D. Mol. Cell. Biol. 1999; 19: 1486-1497Crossref PubMed Google Scholar, 17Corless C.L. Kibel A. Iliopoulos O. Kaelin W.G. Hum. Path. 1997; 28: 459-464Crossref PubMed Scopus (98) Google Scholar, 18Los M. Jansen G.H. Kaelin W.G. Lips C.J.M. Blijham G.H. Voest E.E. Lab. Invest. 1996; 75: 231-238PubMed Google Scholar). The localization of pVHL appears to be regulated according to cell density; pVHL is cytoplasmic in confluent cultures, but shuttles to the nucleus under sparse culture conditions (15Lee S. Chen D.Y.T. Humphrey J.S. Gnarra J.R. Linehan W.M. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1770-1775Crossref PubMed Scopus (135) Google Scholar). Nuclear export of pVHL is reduced by inhibitors of RNA polymerase II and polyadenylation, whereas nuclear import is unaffected, resulting in the localization of pVHL to the nucleus (16Lee S. Neumann M. Stearman R. Stauber R. Pause A. Pavlakis G. Klausner R.D. Mol. Cell. Biol. 1999; 19: 1486-1497Crossref PubMed Google Scholar, 19Groulx I. Bonicalzi M. Lee S. J. Biol. Chem. 2000; 275: 8991-9000Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). VHL has been demonstrated to form a multimeric complex with two components of the transcriptional elongation factor elongin (elongin B and C) (11Iliopoulos O. Ohh M. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11661-11666Crossref PubMed Scopus (205) Google Scholar, 12Schoenfeld A. Davidowitz E. Burk R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8817-8822Crossref PubMed Scopus (186) Google Scholar, 14Duan D.R. Pause A. Burgess W. Aso T. Chen D.Y.T. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (506) Google Scholar, 20Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin Jr., W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (565) Google Scholar), as well as cullin-2 (21Lonergan K. Iliopoulos O. Ohh M. Kamura T. Conaway R.C. Conaway J.W. Kaelin W.G. Mol. Cell. Biol. 1998; 18: 732-741Crossref PubMed Scopus (325) Google Scholar, 22Pause A. Lee S. Worrell R.A. Chen D.Y.T. Burgess W.H. Linehan W.M. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2156-2161Crossref PubMed Scopus (427) Google Scholar) and Rbx1 (23Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin W.G.J. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (660) Google Scholar). Significantly, the majority of VHL mutants are defective in their ability to bind elongin C, implying functional significance for this interaction in vivo (14Duan D.R. Pause A. Burgess W. Aso T. Chen D.Y.T. Garrett K.P. Conaway R.C. Conaway J.W. Linehan W.M. Klausner R.D. Science. 1995; 269: 1402-1406Crossref PubMed Scopus (506) Google Scholar, 20Kibel A. Iliopoulos O. DeCaprio J.A. Kaelin Jr., W.G. Science. 1995; 269: 1444-1446Crossref PubMed Scopus (565) Google Scholar, 24Ohh M. Kaelin Jr., W.G. Mol. Med. Today. 1999; 5: 257-263Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25Stebbins C.E. Kaelin Jr., W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (672) Google Scholar). Additional clues to VHL function were provided by the discovery that elongin C and cullin-2 bear homology to yeast proteins (Skp1 and Cdc53), which function as a ubiquitin E3 ligase when complexed with an F-box protein (26Bai 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 (964) Google Scholar, 27Willems A.R. Lanker S. Patton E.E. Craig K.L. Nason T.F. Mathias N. Kobayashi R. Wittenberg C. Tyers M. Cell. 1996; 86: 453-463Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 28Mathias N. Johnson S.L. Winey M. Adams A.E. Goetsch L. Pringle J.R. Byers B. Goebl M.G. Mol. Cell. Biol. 1996; 16: 6634-6643Crossref PubMed Scopus (182) Google Scholar, 29Jackson P.K. Curr. Biol. 1996; 6: 1209-1212Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Furthermore, anti-pVHL immunoprecipitates can support E3 ubiquitin ligase activity in vitro if supplemented with exogenous ubiquitin-conjugating enzymes (30Iwai 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 (420) Google Scholar, 31Listzwan J. Imbert G. Wirebelauer C. Gstaiger M. Krek W. Genes Dev. 1999; 13: 1822-1833Crossref PubMed Scopus (335) Google Scholar). As a consequence, a model has evolved in which the pVHL-elongin B/C-Cul-2 complex functions as a ubiquitin E3 ligase, which targets specific substrates for ubiquitin-mediated proteasomal degradation (32Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (544) Google Scholar). The absence of pVHL in renal carcinoma cell lines is associated with a hypoxic phenotype under normoxic culture conditions; these cells express increased levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor, and glucose transporter 1 (GLUT-1) (33Iliopoulos O. Jiang C. Levy A.P. Kaelin W.G. Goldberg M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10595-10599Crossref PubMed Scopus (731) Google Scholar,34Gnarra J.R. Zhou S. Merrill M.J. Wagner J.R. Krumm A. Papavassiliou E. Oldfield E.H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10589-10594Crossref PubMed Scopus (464) Google Scholar). The overproduction of these genes likely contributes to the hypervascular phenotype characteristic of VHL disease-associated neoplasms (35Wizigmann-Voos S. Breier G. Risau W. Plate K.H. Cancer Res. 1995; 55: 1358-1364PubMed Google Scholar). The ubiquitin E3 ligase model of pVHL action accounts for the increased transcription of hypoxia-inducible genes (VEGF, platelet-derived growth factor), which occurs under normoxic conditions with pVHL mutations (33Iliopoulos O. Jiang C. Levy A.P. Kaelin W.G. Goldberg M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10595-10599Crossref PubMed Scopus (731) Google Scholar, 34Gnarra J.R. Zhou S. Merrill M.J. Wagner J.R. Krumm A. Papavassiliou E. Oldfield E.H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10589-10594Crossref PubMed Scopus (464) Google Scholar, 36Golde D.W. Hocking W.G. Koeffler H.P. Adamson JW. Ann. Intern. Med. 1981; 95: 71-87Crossref PubMed Scopus (59) Google Scholar, 37Siemeister G. Weindel K. Mohrs K. Barleon B. Martiny-Baron G. Marme D. Cancer Res. 1996; 56: 2299-2301PubMed Google Scholar, 38Stratmann R. Krieg M. Haas R. Plate K.H. J. Neuropathol. Exp. Neurol. 1997; 56: 1242-1252Crossref PubMed Scopus (68) Google Scholar, 39Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar). The transcription of these genes is regulated by the levels of the transcription factor hypoxia-inducible factor (HIF)-1 (Refs. 40Dang C.V. Lewis B.C. Dolde C. Dang G. Shim H. J. Bioenerg. Biomembr. 1997; 29: 345-354Crossref PubMed Scopus (114) Google Scholar and 41Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshert E. Keshet E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2179) Google Scholar, and references therein). Under conditions of normoxia, rapid ubiquitination and proteasomal-dependent degradation of HIF-1α is mediated by an oxygen-dependent degradation domain (42Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1818) Google Scholar). Under hypoxic conditions, the stability of HIF-1α protein increases, resulting in enhanced transcription of hypoxia-inducible genes (43Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (5990) Google Scholar,44Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.Y. Huang L.E. Pavletich N. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1236) Google Scholar). Subsequent studies have shown that pVHL and HIF-1α directly interact under normoxic conditions, resulting in the ubiquitination and proteasomal targeting of HIF-1α by the pVHL-elongin B/C-Cul-2 complex (45Cockman 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 (909) Google Scholar, 46Stein I. Neeman M. Shweiki D. Itin A. Keshet E. Mol. Cell. Biol. 1995; 15: 5363-5368Crossref PubMed Scopus (415) Google Scholar). Thus, in the absence of pVHL or with mutations that alter its ability to function as part of a ubiquitin E3 ligase complex, HIF-1α levels rise, leading to increased transcription of hypoxia-inducible genes such as VEGF (44Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.Y. Huang L.E. Pavletich N. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1236) Google Scholar). Increased GLUT-1 and VEGF mRNA stability has also been observed in cells under conditions of hypoxia (47Maxwell P.H. Wiesener 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 (4022) Google Scholar). In cells lacking pVHL, VEGF mRNA stability has been demonstrated (33Iliopoulos O. Jiang C. Levy A.P. Kaelin W.G. Goldberg M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10595-10599Crossref PubMed Scopus (731) Google Scholar, 34Gnarra J.R. Zhou S. Merrill M.J. Wagner J.R. Krumm A. Papavassiliou E. Oldfield E.H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10589-10594Crossref PubMed Scopus (464) Google Scholar). It has been hypothesized that a similar mechanism accounts for the stabilization of GLUT-1 and VEGF mRNA observed in pVHL-deficient cells (45Cockman 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 (909) Google Scholar, 47Maxwell P.H. Wiesener 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 (4022) Google Scholar). In this model, an RNA-binding protein, instead of a transcription factor such as HIF-1α, constitutes the target by which pVHL regulates the stability of these mRNA. The 3′-UTR of both GLUT-1 and VEGF have been shown to contain AU-rich elements (AURE), which regulate mRNA turnover (48Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3103) Google Scholar). Previous work in our laboratory indicated that hnRNP A2 binds acis-acting instability element in the GLUT-1 3′-UTR that plays a role in the post-transcriptional regulation of GLUT-1 expression (49Hamilton B.J. Nichols R.C. Tsukamoto H. Boado R.J. Pardridge W.M. Rigby W.F. Biochem. Biophys. Res. Commun. 1999; 261: 646-651Crossref PubMed Scopus (73) Google Scholar). These data suggested that overexpression of hnRNP A2 might account for the change in GLUT-1 mRNA turnover associated with the absence of pVHL. In RCC cell lines that differ only in their expression of functional pVHL, we observed that hnRNP A2 expression is increased in pVHL-deficient cell lines. This pVHL-dependent reduction in hnRNP A2 expression occurs independently of cell confluence. Northern blotting demonstrated that hnRNP A2 mRNA levels were unaffected, suggesting that pVHL deficiency results in decreased hnRNP A2 protein expression through changes in protein turnover or translation. Proteasome inhibition of cells expressing wild type pVHL resulted in cytoplasmic accumulation of hnRNP A2, but not the closely related and homologous protein hnRNP A1 (50Burd C.G. Dreyfuss G. EMBO J. 1994; 13: 1197-1204Crossref PubMed Scopus (420) Google Scholar). Finally, anin vivo interaction between pVHL and hnRNP A2 is demonstrated in both proteasomally inhibited cytosolic and untreated nuclear extracts. Collectively, these data indicate that hnRNP A2 expression is regulated by pVHL, and suggest the possibility that hnRNP A2 is a target of the pVHL/elongin B/C/Cul2/Rbx1 ubiquitin degradation machinery. The human 786-0 renal carcinoma cell line (obtained from ATCC) was maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 20% fetal bovine serum (HyClone). 786-0 subclones (generously provided by Dr. William Kaelin) stably transfected with pRc/CMV (pRc-9), pRc/CMV-VHL (C162F), and pRc/CMV-VHL (WT-8) (as previously described in Ref. 10Iliopoulos O. Kibel A. Gray S. Kaelin Jr., W.G. Nat. Med. 1995; 1: 822-826Crossref PubMed Scopus (590) Google Scholar) were cultured in the presence of G418 (500 µg/ml). The UMRC6 (UMRC) human renal carcinoma cell lines (generously provided by Dr. Bert Zbar), stably transfected with either a vector control (parental) or a plasmid expressing wild type VHL cDNA, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For proteasome inhibition experiments, cells were treated with either 100 µm LLnL (Sigma) or 25 µmMG132 (Sigma) overnight. Cytosolic lysates were prepared using a method characterized by its lack of contamination by nuclear proteins (51Hamilton B.J. Nagy E. Malter J.S. Arrick B.A. Rigby W.F. J. Biol. Chem. 1993; 268: 8881-8887Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were washed twice in 1× phosphate-buffered saline, then lysed by gentle resuspension in 1% Triton X-100 lysis buffer (50 µL/2 × 107 cells) consisting of 10 mm PIPES, pH 6.8, 100 mm KCl, 2.5 mm MgCl2, 300 mm sucrose, 1 mm Pefabloc, and 2 µg/ml leupeptin and pepstatin A. Samples were incubated for 3 min on ice and then centrifuged for 5 min at 500 × g. The supernatant was aliquoted and stored at −80 °C as the cytosolic fraction. The pellet was resuspended in lysis buffer and then spun through a 30% sucrose cushion. The nuclear pellet was resuspended in 0.5 nuclear pellet volume in low salt buffer consisting of 10 mm Tris-HCl, pH 7.6, 20 mmKCl, 1.5 mm MgCl2, 0.5 µmdithiothreitol, 0.2 mm EDTA, 25% glycerol, 2 mm Pefabloc, 1 µg/ml each leupeptin and pepstatin A. While vortexing, 1.5 nuclear pellet volume of high salt buffer (containing 0.5 m KCl) was added dropwise. Samples were incubated with agitation for 30 min at 4 °C, and then centrifuged for 30 min at 12,000 × g at 4 °C (52Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9131) Google Scholar). The supernatant was aliquoted and stored at −80 °C as the nuclear fraction. For polysome preparations, pRc-9, C162F, and WT-8 cells were homogenized in buffer A (10 mm Tris-HCl, pH 7.6, 1 mm potassium acetate, 1.5 mm magnesium acetate, 2 mm dithiothreitol, 2 µg/ml leupeptin and pepstatin A, and 2 mm Pefabloc), and nuclei removed by centrifugation. The supernatant was layered over a 30% sucrose cushion followed by ultracentrifugation at 36,000 × rpm for 5 h at 4 °C. Whole cell lysates were prepared by washing cells twice with 1× phosphate-buffered saline, and then solubilizing in 2× Laemmli SDS sample buffer (53Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205506) Google Scholar). Coimmunoprecipitation of pVHL with hnRNP A2 from WT-8 proteasomally inhibited cytoplasmic (100 µg) or untreated nuclear (400 µg) lysates was performed with a mouse monoclonal antibody directed against pVHL (1G32) (10Iliopoulos O. Kibel A. Gray S. Kaelin Jr., W.G. Nat. Med. 1995; 1: 822-826Crossref PubMed Scopus (590) Google Scholar). Immunocomplexes were captured with protein A-Sepharose beads (Amersham Pharmacin) for 2 h at 4 °C, and beads were washed six times in 300 mmNaCl. Proteins were resolved by 15% SDS-PAGE and electrotransferred to nitrocellulose membrane in CAPS buffer, pH 11.0, with 15% methanol. Immunoblots were washed with Tris-buffered saline, 0.1% Tween 20, and blocked in 3% bovine serum albumin overnight at 4 °C. Membranes were then probed with a mouse monoclonal antibody directed against hnRNP A2 (EF67) and goat anti-mouse HRP-conjugated secondary antibody (Bio-Rad). To demonstrate the reciprocal interaction, CNBr-hnRNP A2-conjugated beads were used to immunoprecipitate from WT-8 lysate, and then immunoblotted with 1G32, followed by a goat anti-mouse HRP-conjugated secondary antibody. CNBr beads were used in lieu of protein-A Sepharose in order to eliminate interfering signal generated by detection of the light chain of the secondary antibody. Depleted lysates represent supernatants of immunoprecipitations. Polysomal lysates were probed with a rabbit polyclonal anti-hnRNP A2 antibody (Act2), followed by goat anti-rabbit HRP-conjugated secondary antibody. Detection of hnRNP A1 protein was accomplished by probing with rabbit polyclonal anti-hnRNP A1 antibody (Act1), followed by goat anti-rabbit HRP-conjugated secondary antibody. As indicated, blots were probed with anti-GAPDH monoclonal antibody (6C5-American Research Products), followed by goat anti-mouse HRP-conjugated secondary antibody to control for loading. Reactive antigens were visualized with Supersignal chemiluminescence substrate (Pierce). Total cellular RNA was extracted by acid guanidinium-phenol-chloroform extraction (54Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62898) Google Scholar). RNA was size-fractionated by formaldehyde-agarose gel electrophoresis, blotted onto Hybond-N nylon membrane (Amersham Pharmacia Biotech) in 20× SSC, and baked under vacuum for 2 h at 80 °C. Filters were prehybridized overnight at 42 °C in 50% formamide, 0.8m NaCl, 0.1 m PIPES, 0.1% Sarkosyl, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, and salmon sperm DNA (200 µg/ml). Hybridizations were performed at 42 °C for 24 h in prehybridization mix containing 10% dextran sulfate and 1 × 106 cpm/ml cDNA probes for hnRNP A2 and β2-microglobulin, which had been labeled with [32P]dCTP (3000 Ci/mmol) using a random primer method (55Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16562) Google Scholar). Membranes were washed with 0.1× SSC containing 0.02% sodium pyrophosphate and 0.5% Sarkosyl four times for 30 min each at 56 °C. Blots were dried and exposed at −70 °C to Fuji XAR film using one intensifying screen. Protein and mRNA bands were quantified by densitometric scanning of autoradiographs and immunoblots using NIH Image software. The mechanism by which pVHL deficiency increases GLUT-1 mRNA stability is unknown. It was hypothesized that the effect on GLUT-1 mRNA stability is mediated through a specific GLUT-1 mRNA-binding protein (24Ohh M. Kaelin Jr., W.G. Mol. Med. Today. 1999; 5: 257-263Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Previous work identified hnRNP A2 as a GLUT-1 mRNA 3′-UTR-binding protein (49Hamilton B.J. Nichols R.C. Tsukamoto H. Boado R.J. Pardridge W.M. Rigby W.F. Biochem. Biophys. Res. Commun. 1999; 261: 646-651Crossref PubMed Scopus (73) Google Scholar). For these reasons, we examined hnRNP A2 protein levels in three RCC cell lines, which differ only in their expression of VHL. Cytoplasmic lysates derived from 786-0 RCC cells, which lack functional VHL protein, pRc-9 cells (786-0 cells transfected with empty expression vector), and WT-8 cells (transfected with wild type VHL) were examined for hnRNP A2 expression. Data shown are representative of seven experiments. Immunoblotting demonstrated increased hnRNP A2 levels in cells lacking functional VHL (786-0 and pRc-9) relative to those containing wild type VHL (WT-8) (Fig.1A). Densitometric analysis of hnRNP A2 expression in these lysates demonstrated 3–5-fold elevated levels of this protein in cells lacking wild type pVHL. The change in cytosolic hnRNP A2 levels among the cell lines did not appear to be due to differences in subcellular distribution, as extracts directly lysed in SDS loading buffer (whole cell lysates) and boiled showed similar patterns of expression (Fig. 1 B). The expression of hnRNP A1 and GAPDH was unaffected. This finding indicates that pVHL-dependent regulation of hnRNP A2 was highly specific; hnRNP A1 and hnRNP A2 have 70% homology at the amino acid level (50Burd C.G. Dreyfuss G. EMBO J. 1994; 13: 1197-1204Crossref PubMed Scopus (420) Google Scholar). Consistent with this interpretation, the expression of other AURE-binding proteins reportedly associated with mRNA turnover such as HuR and AUF1 was unaffected (data not shown). Nevertheless, to ensure that the observed hnRNP A2 overexpression observed in the 786-0 and pRc-9 cell lines was not cell line-specific, another set of RCC cell lines was tested (Fig. 1 C). As seen above, UMRC cells transfected with pVHL (WT) exhibited decreased levels of hnRNP A2 relative to the pVHL-deficient parental cell line (PAR). Recent reports have described the influence of cell density on the ability of pVHL to mediate biochemical and morphological differentiation of renal carcinoma cell lines (56Davidowitz E.J. Schoenfeld A.R. Burk R.D. Mol. Cell. Biol. 2001; 21: 865-874Crossref PubMed Scopus (95) Google Scholar, 57Lieubeau-Teillet B. Rak J. Jothy S. Iliopoulos O. Kaelin W. Kerbel R.S. Cancer Res. 1998; 58: 4957-4962PubMed Google Scholar). In these studies, VHL expression promoted renal cell differentiation and growth arrest under conditions of high cell density, in association with down-regulation of integrins and up-regulation of hepatocyte nuclear factor 1α, a global activator of proximal tubule-specific genes (56Davidowitz E.J. Schoenfeld A.R. Burk R.D. Mol. Cell. Biol. 2001; 21: 865-874Crossref PubMed Scopus (95) Google Scholar). To exclude differences in cell density and growth as possible explanations for the disparity in hnRNP A2 protein expression, all of the experiments delineated above were performed with 100% confluent cells. Subsequently, we examined cytosolic levels of hnRNP A2 at various states of confluence (Fig.2). As seen earlier, hnRNP A2 protein was expressed consistently higher in pRc-9 cytosol, independent of cell density. In neither cell line did hnRNP A2 levels vary as a function of cell confluence. Moreover, GAPDH levels are constant between the cell types, regardless of cell density. These data suggest the observed differences in hnRNP A2 protein expression were not a consequence of altered rates of proliferation or differentiation between the cell lines. Some studies indicate that AURE-mediated mRNA turnover requires either polysomal loading or ribosomal transit (58Curatola A.M. Nadal M.S. Schneider R.J. Mol. Cell. Biol. 1995; 15: 6331-6340Crossref PubMed Scopus (69) Google Scholar). Since tot" @default.
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• W2000789117 title "The Von Hippel-Lindau Protein Interacts with Heteronuclear Ribonucleoprotein A2 and Regulates Its Expression" @default.
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