Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Nov 1;127(3):609–622. doi: 10.1083/jcb.127.3.609

The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing

PMCID: PMC2120229  PMID: 7525595

Abstract

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full- length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.

Full Text

The Full Text of this article is available as a PDF (5.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberts A. S., Thorburn A. M., Shenolikar S., Mumby M. C., Feramisco J. R. Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):388–392. doi: 10.1073/pnas.90.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berezney R., Coffey D. S. Nuclear protein matrix: association with newly synthesized DNA. Science. 1975 Jul 25;189(4199):291–293. doi: 10.1126/science.1145202. [DOI] [PubMed] [Google Scholar]
  3. Blencowe B. J., Nickerson J. A., Issner R., Penman S., Sharp P. A. Association of nuclear matrix antigens with exon-containing splicing complexes. J Cell Biol. 1994 Nov;127(3):593–607. doi: 10.1083/jcb.127.3.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bookstein R., Lee W. H. Molecular genetics of the retinoblastoma suppressor gene. Crit Rev Oncog. 1991;2(3):211–227. [PubMed] [Google Scholar]
  5. Breeden L., Nasmyth K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol. 1985;50:643–650. doi: 10.1101/sqb.1985.050.01.078. [DOI] [PubMed] [Google Scholar]
  6. Buchkovich K., Duffy L. A., Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 1989 Sep 22;58(6):1097–1105. doi: 10.1016/0092-8674(89)90508-4. [DOI] [PubMed] [Google Scholar]
  7. Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chatterjee P. K., Flint S. J. Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells. J Virol. 1986 Dec;60(3):1018–1026. doi: 10.1128/jvi.60.3.1018-1026.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen P. L., Chen Y., Shan B., Bookstein R., Lee W. H. Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells. Cell Growth Differ. 1992 Feb;3(2):119–125. [PubMed] [Google Scholar]
  10. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  11. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ciejek E. M., Tsai M. J., O'Malley B. W. Actively transcribed genes are associated with the nuclear matrix. Nature. 1983 Dec 8;306(5943):607–609. doi: 10.1038/306607a0. [DOI] [PubMed] [Google Scholar]
  13. Dalton S., Treisman R. Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell. 1992 Feb 7;68(3):597–612. doi: 10.1016/0092-8674(92)90194-h. [DOI] [PubMed] [Google Scholar]
  14. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  15. DeCaprio J. A., Ludlow J. W., Lynch D., Furukawa Y., Griffin J., Piwnica-Worms H., Huang C. M., Livingston D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. doi: 10.1016/0092-8674(89)90507-2. [DOI] [PubMed] [Google Scholar]
  16. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  17. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  18. Elledge S. J., Mulligan J. T., Ramer S. W., Spottswood M., Davis R. W. Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1731–1735. doi: 10.1073/pnas.88.5.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fu X. D., Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. doi: 10.1038/343437a0. [DOI] [PubMed] [Google Scholar]
  20. Goodrich D. W., Lee W. H. Molecular characterization of the retinoblastoma susceptibility gene. Biochim Biophys Acta. 1993 May 25;1155(1):43–61. doi: 10.1016/0304-419x(93)90021-4. [DOI] [PubMed] [Google Scholar]
  21. Greenfield I., Nickerson J., Penman S., Stanley M. Human papillomavirus 16 E7 protein is associated with the nuclear matrix. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11217–11221. doi: 10.1073/pnas.88.24.11217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  23. Hamel P. A., Cohen B. L., Sorce L. M., Gallie B. L., Phillips R. A. Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions. Mol Cell Biol. 1990 Dec;10(12):6586–6595. doi: 10.1128/mcb.10.12.6586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. He D. C., Nickerson J. A., Penman S. Core filaments of the nuclear matrix. J Cell Biol. 1990 Mar;110(3):569–580. doi: 10.1083/jcb.110.3.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Helin K., Lees J. A., Vidal M., Dyson N., Harlow E., Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. doi: 10.1016/0092-8674(92)90107-n. [DOI] [PubMed] [Google Scholar]
  26. Hensey C. E., Hong F., Durfee T., Qian Y. W., Lee E. Y., Lee W. H. Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization. J Biol Chem. 1994 Jan 14;269(2):1380–1387. [PubMed] [Google Scholar]
  27. Hinds P. W., Mittnacht S., Dulic V., Arnold A., Reed S. I., Weinberg R. A. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992 Sep 18;70(6):993–1006. doi: 10.1016/0092-8674(92)90249-c. [DOI] [PubMed] [Google Scholar]
  28. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  29. Hogg A., Bia B., Onadim Z., Cowell J. K. Molecular mechanisms of oncogenic mutations in tumors from patients with bilateral and unilateral retinoblastoma. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7351–7355. doi: 10.1073/pnas.90.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Huang S., Spector D. L. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. doi: 10.1101/gad.5.12a.2288. [DOI] [PubMed] [Google Scholar]
  31. Huang S., Wang N. P., Tseng B. Y., Lee W. H., Lee E. H. Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. EMBO J. 1990 Jun;9(6):1815–1822. doi: 10.1002/j.1460-2075.1990.tb08306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Imai Y., Matsushima Y., Sugimura T., Terada M. Purification and characterization of human papillomavirus type 16 E7 protein with preferential binding capacity to the underphosphorylated form of retinoblastoma gene product. J Virol. 1991 Sep;65(9):4966–4972. doi: 10.1128/jvi.65.9.4966-4972.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jackson D. A., Hassan A. B., Errington R. J., Cook P. R. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. doi: 10.1002/j.1460-2075.1993.tb05747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kaelin W. G., Jr, Ewen M. E., Livingston D. M. Definition of the minimal simian virus 40 large T antigen- and adenovirus E1A-binding domain in the retinoblastoma gene product. Mol Cell Biol. 1990 Jul;10(7):3761–3769. doi: 10.1128/mcb.10.7.3761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  36. Kaelin W. G., Jr, Pallas D. C., DeCaprio J. A., Kaye F. J., Livingston D. M. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell. 1991 Feb 8;64(3):521–532. doi: 10.1016/0092-8674(91)90236-r. [DOI] [PubMed] [Google Scholar]
  37. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lee W. H., Hollingsworth R. E., Jr, Qian Y. W., Chen P. L., Hong F., Lee E. Y. RB protein as a cellular "corral" for growth-promoting proteins. Cold Spring Harb Symp Quant Biol. 1991;56:211–217. doi: 10.1101/sqb.1991.056.01.026. [DOI] [PubMed] [Google Scholar]
  39. Lee W. H., Shew J. Y., Hong F. D., Sery T. W., Donoso L. A., Young L. J., Bookstein R., Lee E. Y. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity. Nature. 1987 Oct 15;329(6140):642–645. doi: 10.1038/329642a0. [DOI] [PubMed] [Google Scholar]
  40. Lees J. A., Buchkovich K. J., Marshak D. R., Anderson C. W., Harlow E. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J. 1991 Dec;10(13):4279–4290. doi: 10.1002/j.1460-2075.1991.tb05006.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lin B. T., Gruenwald S., Morla A. O., Lee W. H., Wang J. Y. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase. EMBO J. 1991 Apr;10(4):857–864. doi: 10.1002/j.1460-2075.1991.tb08018.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ludlow J. W., DeCaprio J. A., Huang C. M., Lee W. H., Paucha E., Livingston D. M. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell. 1989 Jan 13;56(1):57–65. doi: 10.1016/0092-8674(89)90983-5. [DOI] [PubMed] [Google Scholar]
  43. Ludlow J. W., Glendening C. L., Livingston D. M., DeCarprio J. A. Specific enzymatic dephosphorylation of the retinoblastoma protein. Mol Cell Biol. 1993 Jan;13(1):367–372. doi: 10.1128/mcb.13.1.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mancini M. A., Shan B., Nickerson J. A., Penman S., Lee W. H. The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):418–422. doi: 10.1073/pnas.91.1.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mittnacht S., Weinberg R. A. G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment. Cell. 1991 May 3;65(3):381–393. doi: 10.1016/0092-8674(91)90456-9. [DOI] [PubMed] [Google Scholar]
  46. Nickerson J. A., Penman S. Localization of nuclear matrix core filament proteins at interphase and mitosis. Cell Biol Int Rep. 1992 Aug;16(8):811–826. doi: 10.1016/s0309-1651(05)80024-4. [DOI] [PubMed] [Google Scholar]
  47. Qian Y., Luckey C., Horton L., Esser M., Templeton D. J. Biological function of the retinoblastoma protein requires distinct domains for hyperphosphorylation and transcription factor binding. Mol Cell Biol. 1992 Dec;12(12):5363–5372. doi: 10.1128/mcb.12.12.5363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Qin X. Q., Chittenden T., Livingston D. M., Kaelin W. G., Jr Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 1992 Jun;6(6):953–964. doi: 10.1101/gad.6.6.953. [DOI] [PubMed] [Google Scholar]
  49. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  50. Shan B., Zhu X., Chen P. L., Durfee T., Yang Y., Sharp D., Lee W. H. Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. Mol Cell Biol. 1992 Dec;12(12):5620–5631. doi: 10.1128/mcb.12.12.5620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shew J. Y., Ling N., Yang X. M., Fodstad O., Lee W. H. Antibodies detecting abnormalities of the retinoblastoma susceptibility gene product (pp110RB) in osteosarcomas and synovial sarcomas. Oncogene Res. 1989;4(3):205–214. [PubMed] [Google Scholar]
  52. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  53. Spector D. L. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. doi: 10.1146/annurev.cb.09.110193.001405. [DOI] [PubMed] [Google Scholar]
  54. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  55. Szekely L., Uzvolgyi E., Jiang W. Q., Durko M., Wiman K. G., Klein G., Sumegi J. Subcellular localization of the retinoblastoma protein. Cell Growth Differ. 1991 Jun;2(6):287–295. [PubMed] [Google Scholar]
  56. Templeton D. J. Nuclear binding of purified retinoblastoma gene product is determined by cell cycle-regulated phosphorylation. Mol Cell Biol. 1992 Feb;12(2):435–443. doi: 10.1128/mcb.12.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Templeton D. J., Park S. H., Lanier L., Weinberg R. A. Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3033–3037. doi: 10.1073/pnas.88.8.3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wan K. M., Nickerson J. A., Krockmalnic G., Penman S. The B1C8 protein is in the dense assemblies of the nuclear matrix and relocates to the spindle and pericentriolar filaments at mitosis. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):594–598. doi: 10.1073/pnas.91.2.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wansink D. G., Schul W., van der Kraan I., van Steensel B., van Driel R., de Jong L. Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol. 1993 Jul;122(2):283–293. doi: 10.1083/jcb.122.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]
  61. Xing Y., Johnson C. V., Dobner P. R., Lawrence J. B. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. doi: 10.1126/science.8446901. [DOI] [PubMed] [Google Scholar]
  62. Xing Y., Lawrence J. B. Nuclear RNA tracks: structural basis for transcription and splicing? Trends Cell Biol. 1993 Oct;3(10):346–353. doi: 10.1016/0962-8924(93)90105-a. [DOI] [PubMed] [Google Scholar]
  63. Yang X., Hubbard E. J., Carlson M. A protein kinase substrate identified by the two-hybrid system. Science. 1992 Jul 31;257(5070):680–682. doi: 10.1126/science.1496382. [DOI] [PubMed] [Google Scholar]
  64. Zeitlin S., Wilson R. C., Efstratiadis A. Autonomous splicing and complementation of in vivo-assembled spliceosomes. J Cell Biol. 1989 Mar;108(3):765–777. doi: 10.1083/jcb.108.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES