Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jan 4;91(1):418–422. doi: 10.1073/pnas.91.1.418

The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein.

M A Mancini 1, B Shan 1, J A Nickerson 1, S Penman 1, W H Lee 1
PMCID: PMC42959  PMID: 8278403

Abstract

The retinoblastoma gene product (Rb) has been established as a tumor suppressor and cell cycle regulator, although its mechanism of action remains obscure. The observations that several Rb-binding viral oncoproteins all associate with the nuclear matrix suggest that these interactions may occur on this structure. To determine whether Rb itself is a component of the matrix, we extracted synchronized cultured cells to isolate matrix proteins while preserving nuclear architecture. Immunoblot and immunolabeling data show that a significant portion of hypophosphorylated Rb associates with the matrix only during early G1. Mutant Rb in tumor cells did not associate with the matrix, whereas Rb-reconstituted cells contained abundant matrix-bound Rb. Rb is distributed widely throughout the matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants. Core filaments of the matrix contained no detectable Rb. Our screening of expression libraries for potential Rb-associated proteins has identified several that are part of the matrix. Specifically, the peripheral matrix proteins lamin A and C bound Rb in vitro. We therefore suggest that Rb interactions with the nuclear matrix may be important for its ability to regulate cell cycle progression.

Full text

PDF
418

Images in this article

419Fig. 1
on p.419
419Fig. 2
on p.419
419Fig. 3
on p.419
420Fig. 4
on p.420
421Fig. 5
on p.421
421Fig. 6
on p.421

Selected References

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

  1. 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]
  2. Bidwell J. P., Van Wijnen A. J., Fey E. G., Dworetzky S., Penman S., Stein J. L., Lian J. B., Stein G. S. Osteocalcin gene promoter-binding factors are tissue-specific nuclear matrix components. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3162–3166. doi: 10.1073/pnas.90.8.3162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bookstein R., Shew J. Y., Chen P. L., Scully P., Lee W. H. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science. 1990 Feb 9;247(4943):712–715. doi: 10.1126/science.2300823. [DOI] [PubMed] [Google Scholar]
  4. Bridger J. M., Kill I. R., O'Farrell M., Hutchison C. J. Internal lamin structures within G1 nuclei of human dermal fibroblasts. J Cell Sci. 1993 Feb;104(Pt 2):297–306. doi: 10.1242/jcs.104.2.297. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Defeo-Jones D., Huang P. S., Jones R. E., Haskell K. M., Vuocolo G. A., Hanobik M. G., Huber H. E., Oliff A. Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product. Nature. 1991 Jul 18;352(6332):251–254. doi: 10.1038/352251a0. [DOI] [PubMed] [Google Scholar]
  10. Deppert W., Von Der Weth A. Functional interaction of nuclear transport-defective simian virus 40 large T antigen with chromatin and nuclear matrix. J Virol. 1990 Feb;64(2):838–846. doi: 10.1128/jvi.64.2.838-846.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Fey E. G., Krochmalnic G., Penman S. The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J Cell Biol. 1986 May;102(5):1654–1665. doi: 10.1083/jcb.102.5.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Getzenberg R. H., Pienta K. J., Coffey D. S. The tissue matrix: cell dynamics and hormone action. Endocr Rev. 1990 Aug;11(3):399–417. doi: 10.1210/edrv-11-3-399. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Goodrich D. W., Wang N. P., Qian Y. W., Lee E. Y., Lee W. H. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell. 1991 Oct 18;67(2):293–302. doi: 10.1016/0092-8674(91)90181-w. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Huang H. J., Yee J. K., Shew J. Y., Chen P. L., Bookstein R., Friedmann T., Lee E. Y., Lee W. H. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science. 1988 Dec 16;242(4885):1563–1566. doi: 10.1126/science.3201247. [DOI] [PubMed] [Google Scholar]
  21. Huang S., Lee W. H., Lee E. Y. A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product. Nature. 1991 Mar 14;350(6314):160–162. doi: 10.1038/350160a0. [DOI] [PubMed] [Google Scholar]
  22. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Keyomarsi K., Sandoval L., Band V., Pardee A. B. Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res. 1991 Jul 1;51(13):3602–3609. [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. McCready S. J., Godwin J., Mason D. W., Brazell I. A., Cook P. R. DNA is replicated at the nuclear cage. J Cell Sci. 1980 Dec;46:365–386. doi: 10.1242/jcs.46.1.365. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Nickerson J. A., Krockmalnic G., He D. C., Penman S. Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2259–2263. doi: 10.1073/pnas.87.6.2259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pardoll D. M., Vogelstein B., Coffey D. S. A fixed site of DNA replication in eucaryotic cells. Cell. 1980 Feb;19(2):527–536. doi: 10.1016/0092-8674(80)90527-9. [DOI] [PubMed] [Google Scholar]
  31. Pienta K. J., Coffey D. S. Cellular harmonic information transfer through a tissue tensegrity-matrix system. Med Hypotheses. 1991 Jan;34(1):88–95. doi: 10.1016/0306-9877(91)90072-7. [DOI] [PubMed] [Google Scholar]
  32. Rustgi A. K., Dyson N., Bernards R. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product. Nature. 1991 Aug 8;352(6335):541–544. doi: 10.1038/352541a0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Takahashi R., Hashimoto T., Xu H. J., Hu S. X., Matsui T., Miki T., Bigo-Marshall H., Aaronson S. A., Benedict W. F. The retinoblastoma gene functions as a growth and tumor suppressor in human bladder carcinoma cells. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5257–5261. doi: 10.1073/pnas.88.12.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Xing Y. G., Lawrence J. B. Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol. 1991 Mar;112(6):1055–1063. doi: 10.1083/jcb.112.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zeitlin S., Parent A., Silverstein S., Efstratiadis A. Pre-mRNA splicing and the nuclear matrix. Mol Cell Biol. 1987 Jan;7(1):111–120. doi: 10.1128/mcb.7.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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 Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES