Abstract
Simian virus 40 large T-antigen (TAg) transformation is thought to be mediated, at least in part, by binding to and modulating the function of certain cellular proteins, including the retinoblastoma tumor suppressor gene product, pRb. TAg can disrupt the inhibitory complexes formed by pRb with the oncogenic transcription factor E2F, and this mechanism has been suggested to be important for TAg-mediated transformation. Residues 102 to 114 of TAg (including the LXCXE motif) are required for binding to pRb. Mutations within this LXCXE motif abolish the ability of TAg to bind to pRb as well as to transform certain cell types. TAg can also bind to at least two other cellular proteins, p107 and p130, that are related to pRb by sequence homology and share the ability to bind E2F. However, whether p107 and p130 are also targets in TAg-mediated transformation is less clear. To assess the relative contribution of the inactivation of pRb, p107, and p130 to transformation by TAg, fibroblasts were prepared from embryos derived from matings of mice heterozygous for an Rb knockout allele. The ability of TAg to transform fibroblasts homozygous for either wild-type or knockout Rb alleles was evaluated. It is demonstrated that the integrity of the LXCXE motif provides a growth advantage in Rb+/+ and Rb-/- cells. Furthermore, wild-type TAg, but not the LXCXE mutants, could bind to p107 and p130 and disrupt p107-E2F and p130-E2F binding complexes. These results suggest that p107 and p130 participate in TAg-mediated transformation and that they may behave as tumor suppressors.
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- Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
- Brown M., McCormack M., Zinn K. G., Farrell M. P., Bikel I., Livingston D. M. A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth. J Virol. 1986 Oct;60(1):290–293. doi: 10.1128/jvi.60.1.290-293.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Chen S., Paucha E. Identification of a region of simian virus 40 large T antigen required for cell transformation. J Virol. 1990 Jul;64(7):3350–3357. doi: 10.1128/jvi.64.7.3350-3357.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chittenden T., Livingston D. M., DeCaprio J. A. Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of free E2F. Mol Cell Biol. 1993 Jul;13(7):3975–3983. doi: 10.1128/mcb.13.7.3975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clarke A. R., Maandag E. R., van Roon M., van der Lugt N. M., van der Valk M., Hooper M. L., Berns A., te Riele H. Requirement for a functional Rb-1 gene in murine development. Nature. 1992 Sep 24;359(6393):328–330. doi: 10.1038/359328a0. [DOI] [PubMed] [Google Scholar]
- Claudio P. P., Howard C. M., Baldi A., De Luca A., Fu Y., Condorelli G., Sun Y., Colburn N., Calabretta B., Giordano A. p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107. Cancer Res. 1994 Nov 1;54(21):5556–5560. [PubMed] [Google Scholar]
- Cobrinik D., Whyte P., Peeper D. S., Jacks T., Weinberg R. A. Cell cycle-specific association of E2F with the p130 E1A-binding protein. Genes Dev. 1993 Dec;7(12A):2392–2404. doi: 10.1101/gad.7.12a.2392. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Dyson N., Dembski M., Fattaey A., Ngwu C., Ewen M., Helin K. Analysis of p107-associated proteins: p107 associates with a form of E2F that differs from pRB-associated E2F-1. J Virol. 1993 Dec;67(12):7641–7647. doi: 10.1128/jvi.67.12.7641-7647.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ewen M. E., Ludlow J. W., Marsilio E., DeCaprio J. A., Millikan R. C., Cheng S. H., Paucha E., Livingston D. M. An N-terminal transformation-governing sequence of SV40 large T antigen contributes to the binding of both p110Rb and a second cellular protein, p120. Cell. 1989 Jul 28;58(2):257–267. doi: 10.1016/0092-8674(89)90840-4. [DOI] [PubMed] [Google Scholar]
- Ewen M. E., Sluss H. K., Sherr C. J., Matsushime H., Kato J., Livingston D. M. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell. 1993 May 7;73(3):487–497. doi: 10.1016/0092-8674(93)90136-e. [DOI] [PubMed] [Google Scholar]
- Ewen M. E., Xing Y. G., Lawrence J. B., Livingston D. M. Molecular cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene product-related protein. Cell. 1991 Sep 20;66(6):1155–1164. doi: 10.1016/0092-8674(91)90038-z. [DOI] [PubMed] [Google Scholar]
- Figge J., Webster T., Smith T. F., Paucha E. Prediction of similar transforming regions in simian virus 40 large T, adenovirus E1A, and myc oncoproteins. J Virol. 1988 May;62(5):1814–1818. doi: 10.1128/jvi.62.5.1814-1818.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ginsberg D., Vairo G., Chittenden T., Xiao Z. X., Xu G., Wydner K. L., DeCaprio J. A., Lawrence J. B., Livingston D. M. E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev. 1994 Nov 15;8(22):2665–2679. doi: 10.1101/gad.8.22.2665. [DOI] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Harlow E., Franza B. R., Jr, Schley C. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products. J Virol. 1985 Sep;55(3):533–546. doi: 10.1128/jvi.55.3.533-546.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harlow E., Whyte P., Franza B. R., Jr, Schley C. Association of adenovirus early-region 1A proteins with cellular polypeptides. Mol Cell Biol. 1986 May;6(5):1579–1589. doi: 10.1128/mcb.6.5.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Hiebert S. W., Lipp M., Nevins J. R. E1A-dependent trans-activation of the human MYC promoter is mediated by the E2F factor. Proc Natl Acad Sci U S A. 1989 May;86(10):3594–3598. doi: 10.1073/pnas.86.10.3594. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Horowitz J. M., Yandell D. W., Park S. H., Canning S., Whyte P., Buchkovich K., Harlow E., Weinberg R. A., Dryja T. P. Point mutational inactivation of the retinoblastoma antioncogene. Science. 1989 Feb 17;243(4893):937–940. doi: 10.1126/science.2521957. [DOI] [PubMed] [Google Scholar]
- Hu Q. J., Bautista C., Edwards G. M., Defeo-Jones D., Jones R. E., Harlow E. Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides. Mol Cell Biol. 1991 Nov;11(11):5792–5799. doi: 10.1128/mcb.11.11.5792. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacks T., Fazeli A., Schmitt E. M., Bronson R. T., Goodell M. A., Weinberg R. A. Effects of an Rb mutation in the mouse. Nature. 1992 Sep 24;359(6393):295–300. doi: 10.1038/359295a0. [DOI] [PubMed] [Google Scholar]
- Johnson D. G., Cress W. D., Jakoi L., Nevins J. R. Oncogenic capacity of the E2F1 gene. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12823–12827. doi: 10.1073/pnas.91.26.12823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson D. G., Ohtani K., Nevins J. R. Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev. 1994 Jul 1;8(13):1514–1525. doi: 10.1101/gad.8.13.1514. [DOI] [PubMed] [Google Scholar]
- 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]
- Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
- Kimelman D., Miller J. S., Porter D., Roberts B. E. E1a regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related. J Virol. 1985 Feb;53(2):399–409. doi: 10.1128/jvi.53.2.399-409.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lam E. W., Watson R. J. An E2F-binding site mediates cell-cycle regulated repression of mouse B-myb transcription. EMBO J. 1993 Jul;12(7):2705–2713. doi: 10.1002/j.1460-2075.1993.tb05932.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee E. Y., Chang C. Y., Hu N., Wang Y. C., Lai C. C., Herrup K., Lee W. H., Bradley A. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature. 1992 Sep 24;359(6393):288–294. doi: 10.1038/359288a0. [DOI] [PubMed] [Google Scholar]
- Lees J. A., Saito M., Vidal M., Valentine M., Look T., Harlow E., Dyson N., Helin K. The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol. 1993 Dec;13(12):7813–7825. doi: 10.1128/mcb.13.12.7813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li Y., Graham C., Lacy S., Duncan A. M., Whyte P. The adenovirus E1A-associated 130-kD protein is encoded by a member of the retinoblastoma gene family and physically interacts with cyclins A and E. Genes Dev. 1993 Dec;7(12A):2366–2377. doi: 10.1101/gad.7.12a.2366. [DOI] [PubMed] [Google Scholar]
- Livingston D. M., Bradley M. K. The simian virus 40 large T antigen. A lot packed into a little. Mol Biol Med. 1987 Apr;4(2):63–80. [PubMed] [Google Scholar]
- 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]
- Ludlow J. W., Shon J., Pipas J. M., Livingston D. M., DeCaprio J. A. The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell. 1990 Feb 9;60(3):387–396. doi: 10.1016/0092-8674(90)90590-b. [DOI] [PubMed] [Google Scholar]
- Marsilio E., Cheng S. H., Schaffhausen B., Paucha E., Livingston D. M. The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence. J Virol. 1991 Oct;65(10):5647–5652. doi: 10.1128/jvi.65.10.5647-5652.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mayol X., Graña X., Baldi A., Sang N., Hu Q., Giordano A. Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain. Oncogene. 1993 Sep;8(9):2561–2566. [PubMed] [Google Scholar]
- Moran E. A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus E1A products. Nature. 1988 Jul 14;334(6178):168–170. doi: 10.1038/334168a0. [DOI] [PubMed] [Google Scholar]
- Mudryj M., Hiebert S. W., Nevins J. R. A role for the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway. EMBO J. 1990 Jul;9(7):2179–2184. doi: 10.1002/j.1460-2075.1990.tb07387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neuman E., Flemington E. K., Sellers W. R., Kaelin W. G., Jr Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter. Mol Cell Biol. 1994 Oct;14(10):6607–6615. doi: 10.1128/mcb.14.10.6607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nigro J. M., Baker S. J., Preisinger A. C., Jessup J. M., Hostetter R., Cleary K., Bigner S. H., Davidson N., Baylin S., Devilee P. Mutations in the p53 gene occur in diverse human tumour types. Nature. 1989 Dec 7;342(6250):705–708. doi: 10.1038/342705a0. [DOI] [PubMed] [Google Scholar]
- Nossal G. J. The molecular and cellular basis of affinity maturation in the antibody response. Cell. 1992 Jan 10;68(1):1–2. doi: 10.1016/0092-8674(92)90198-l. [DOI] [PubMed] [Google Scholar]
- Peden K. W., Srinivasan A., Farber J. M., Pipas J. M. Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53. Virology. 1989 Jan;168(1):13–21. doi: 10.1016/0042-6822(89)90398-x. [DOI] [PubMed] [Google Scholar]
- Phelps W. C., Münger K., Yee C. L., Barnes J. A., Howley P. M. Structure-function analysis of the human papillomavirus type 16 E7 oncoprotein. J Virol. 1992 Apr;66(4):2418–2427. doi: 10.1128/jvi.66.4.2418-2427.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raychaudhuri P., Rooney R., Nevins J. R. Identification of an E1A-inducible cellular factor that interacts with regulatory sequences within the adenovirus E4 promoter. EMBO J. 1987 Dec 20;6(13):4073–4081. doi: 10.1002/j.1460-2075.1987.tb02753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwarz J. K., Devoto S. H., Smith E. J., Chellappan S. P., Jakoi L., Nevins J. R. Interactions of the p107 and Rb proteins with E2F during the cell proliferation response. EMBO J. 1993 Mar;12(3):1013–1020. doi: 10.1002/j.1460-2075.1993.tb05742.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Singh P., Wong S. H., Hong W. Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation. EMBO J. 1994 Jul 15;13(14):3329–3338. doi: 10.1002/j.1460-2075.1994.tb06635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- TODARO G. J., GREEN H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963 May;17:299–313. doi: 10.1083/jcb.17.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tevethia M. J., Pipas J. M., Kierstead T., Cole C. Requirements for immortalization of primary mouse embryo fibroblasts probed with mutants bearing deletions in the 3' end of SV40 gene A. Virology. 1988 Jan;162(1):76–89. doi: 10.1016/0042-6822(88)90396-0. [DOI] [PubMed] [Google Scholar]
- Thompson D. L., Kalderon D., Smith A. E., Tevethia M. J. Dissociation of Rb-binding and anchorage-independent growth from immortalization and tumorigenicity using SV40 mutants producing N-terminally truncated large T antigens. Virology. 1990 Sep;178(1):15–34. doi: 10.1016/0042-6822(90)90375-2. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vairo G., Livingston D. M., Ginsberg D. Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members. Genes Dev. 1995 Apr 1;9(7):869–881. doi: 10.1101/gad.9.7.869. [DOI] [PubMed] [Google Scholar]
- Weinberg R. A. The retinoblastoma gene and gene product. Cancer Surv. 1992;12:43–57. [PubMed] [Google Scholar]
- Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
- Xu G., Livingston D. M., Krek W. Multiple members of the E2F transcription factor family are the products of oncogenes. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1357–1361. doi: 10.1073/pnas.92.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhu J. Y., Abate M., Rice P. W., Cole C. N. The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53. J Virol. 1991 Dec;65(12):6872–6880. doi: 10.1128/jvi.65.12.6872-6880.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhu L., van den Heuvel S., Helin K., Fattaey A., Ewen M., Livingston D., Dyson N., Harlow E. Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. Genes Dev. 1993 Jul;7(7A):1111–1125. doi: 10.1101/gad.7.7a.1111. [DOI] [PubMed] [Google Scholar]