Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1982 Oct;2(10):1187–1198. doi: 10.1128/mcb.2.10.1187

Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity.

B S Schaffhausen, H Dorai, G Arakere, T L Benjamin
PMCID: PMC369917  PMID: 6184609

Abstract

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.

Full text

PDF
1187

Images in this article

1190Image
on p.1190
1191Image
on p.1191
1192Image
on p.1192
1192Image
on p.1192
1193Image
on p.1193
1193Image
on p.1193
1194Image
on p.1194
1194Image
on p.1194
1194Image
on p.1194
1195Image
on p.1195
1195Image
on p.1195

Selected References

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

  1. Andres R. Y., Jeng I., Bradshaw R. A. Nerve growth factor receptors: identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root neurons. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2785–2789. doi: 10.1073/pnas.74.7.2785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  3. Bendig M. M., Thomas T., Folk W. R. Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome. J Virol. 1980 Mar;33(3):1215–1220. doi: 10.1128/jvi.33.3.1215-1220.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benjamin T. L., Burger M. M. Absence of a cell membrane alteration function in non-transforming mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Oct;67(2):929–934. doi: 10.1073/pnas.67.2.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benjamin T. L., Carmichael G. G., Schaffhausen B. S. The hr-t gene of polyoma virus. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):263–270. doi: 10.1101/sqb.1980.044.01.030. [DOI] [PubMed] [Google Scholar]
  6. Benjamin T. L. Host range mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Sep;67(1):394–399. doi: 10.1073/pnas.67.1.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Benjamin T. L., Schaffhausen B. S., Silver J. E. Polyoma T (tumor) antigen species in abortively and stably transformed cells. J Supramol Struct. 1979;12(1):127–137. doi: 10.1002/jss.400120110. [DOI] [PubMed] [Google Scholar]
  8. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  9. Brown S., Levinson W., Spudich J. A. Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J Supramol Struct. 1976;5(2):119–130. doi: 10.1002/jss.400050203. [DOI] [PubMed] [Google Scholar]
  10. Burr J. G., Dreyfuss G., Penman S., Buchanan J. M. Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3484–3488. doi: 10.1073/pnas.77.6.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. COOPERSTEIN S. J., LAZAROW A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951 Apr;189(2):665–670. [PubMed] [Google Scholar]
  12. Carmichael G. G., Schaffhausen B. S., Dorsky D. I., Oliver D. B., Benjamin T. L. Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3579–3583. doi: 10.1073/pnas.79.11.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Clertant P., Cuzin F. Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses. J Biol Chem. 1982 Jun 10;257(11):6300–6305. [PubMed] [Google Scholar]
  14. Cohen C. M., Kalish D. I., Jacobson B. S., Branton D. Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells. J Cell Biol. 1977 Oct;75(1):119–134. doi: 10.1083/jcb.75.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Easterbrook-Smith S. B., Wallace J. C., Keech D. B. Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site. Eur J Biochem. 1976 Feb 2;62(1):125–130. doi: 10.1111/j.1432-1033.1976.tb10105.x. [DOI] [PubMed] [Google Scholar]
  16. Eckhart W., Hutchinson M. A., Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. doi: 10.1016/0092-8674(79)90205-8. [DOI] [PubMed] [Google Scholar]
  17. Erikson E., Collett M. S., Erikson R. L. In vitro synthesis of a functional avian sarcoma virus transforming-gene product. Nature. 1978 Aug 31;274(5674):919–921. doi: 10.1038/274919a0. [DOI] [PubMed] [Google Scholar]
  18. Erikson R. L., Collett M. S., Erikson E., Purchio A. F. Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6260–6264. doi: 10.1073/pnas.76.12.6260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Feldman R. A., Hanafusa T., Hanafusa H. Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus. Cell. 1980 Dec;22(3):757–765. doi: 10.1016/0092-8674(80)90552-8. [DOI] [PubMed] [Google Scholar]
  20. Feunteun J., Sompayrac L., Fluck M., Benjamin T. Localization of gene functions in polyoma virus DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4169–4173. doi: 10.1073/pnas.73.11.4169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gaudray P., Clertant P., Cuzin F. ATP phosphohydrolase (ATPase) activity of a polyoma virus T antigen. Eur J Biochem. 1980 Aug;109(2):553–560. doi: 10.1111/j.1432-1033.1980.tb04827.x. [DOI] [PubMed] [Google Scholar]
  22. Gilmer T. M., Erikson R. L. Rous sarcoma virus transforming protein, p60src, expressed in E. coli, functions as a protein kinase. Nature. 1981 Dec 24;294(5843):771–773. doi: 10.1038/294771a0. [DOI] [PubMed] [Google Scholar]
  23. Goldfine I. D., Smith G. J. Binding of insulin to isolated nuclei. Proc Natl Acad Sci U S A. 1976 May;73(5):1427–1431. doi: 10.1073/pnas.73.5.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Graham J. M., Hynes R. O., Davidson E. A., Bainton D. F. The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast. Cell. 1975 Apr;4(4):353–365. doi: 10.1016/0092-8674(75)90156-7. [DOI] [PubMed] [Google Scholar]
  25. Griffin B. E., Maddock C. New classes of viable deletion mutants in the early region of polyoma virus. J Virol. 1979 Sep;31(3):645–656. doi: 10.1128/jvi.31.3.645-656.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Haley B. E., Hoffman J. F. Interactions of a photo-affinity ATP analog with cation-stimulated adenosine triphosphatases of human red cell membranes. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3367–3371. doi: 10.1073/pnas.71.9.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hoppe J., Freist W. Localization of the high-affinity ATP site in adenosine-3':5'-monophosphate-dependent protein kinase type I. Photoaffinity labelling studies with 8-azidoadenosine 5'-triphosphate. Eur J Biochem. 1979 Jan 2;93(1):141–146. doi: 10.1111/j.1432-1033.1979.tb12804.x. [DOI] [PubMed] [Google Scholar]
  28. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hutchinson M. A., Hunter T., Eckhart W. Characterization of T antigens in polyoma-infected and transformed cells. Cell. 1978 Sep;15(1):65–77. doi: 10.1016/0092-8674(78)90083-1. [DOI] [PubMed] [Google Scholar]
  30. Ito Y., Brocklehurst J. R., Dulbecco R. Virus-specific proteins in the plasma membrane of cells lytically infected or transformed by pol-oma virus. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4666–4670. doi: 10.1073/pnas.74.10.4666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ito Y. Polyoma virus-specific 55K protein isolated from plasma membrane of productively infected cells is virus-coded and important for cell transformation. Virology. 1979 Oct 15;98(1):261–266. doi: 10.1016/0042-6822(79)90545-2. [DOI] [PubMed] [Google Scholar]
  32. Jacobson B. S. Imporved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum. Biochim Biophys Acta. 1980 Aug 14;600(3):769–780. doi: 10.1016/0005-2736(80)90479-4. [DOI] [PubMed] [Google Scholar]
  33. Jorgensen P. L. Purification and characterization of (Na+ + K+)-ATPase. VI. Differential tryptic modification of catalytic functions of the purified enzyme in presence of NaCl and KCl. Biochim Biophys Acta. 1977 Apr 1;466(1):97–108. doi: 10.1016/0005-2736(77)90211-5. [DOI] [PubMed] [Google Scholar]
  34. Jorgensen P. L. Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool. Biochim Biophys Acta. 1975 Sep 2;401(3):399–415. doi: 10.1016/0005-2736(75)90239-4. [DOI] [PubMed] [Google Scholar]
  35. Jørgensen P. L., Klodos I. Purification and characterization of (Na+ + K+)-ATPase. VII. Tryptic degradation of the Na-form of the enzyme protein resulting in selective modification of dephosphorylation reactions of the (Na+ + K+)-ATPase. Biochim Biophys Acta. 1978 Feb 2;507(1):8–16. doi: 10.1016/0005-2736(78)90369-3. [DOI] [PubMed] [Google Scholar]
  36. Kawai S., Yoshida M., Segawa K., Sugiyama H., Ishizaki R., Toyoshima K. Characterization of Y73, an avian sarcoma virus: a unique transforming gene and its product, a phosphopolyprotein with protein kinase activity. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6199–6203. doi: 10.1073/pnas.77.10.6199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kreil G. Transfer of proteins across membranes. Annu Rev Biochem. 1981;50:317–348. doi: 10.1146/annurev.bi.50.070181.001533. [DOI] [PubMed] [Google Scholar]
  38. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  39. Lania L., Griffiths M., Cooke B., Ito Y., Fried M. Untransformed rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t) mutant. Cell. 1979 Nov;18(3):793–802. doi: 10.1016/0092-8674(79)90132-6. [DOI] [PubMed] [Google Scholar]
  40. Levinson A. D., Oppermann H., Varmus H. E., Bishop J. M. The purified product of the transforming gene of avian sarcoma virus phosphorylates tyrosine. J Biol Chem. 1980 Dec 25;255(24):11973–11980. [PubMed] [Google Scholar]
  41. Magnusson G., Berg P. Construction and analysis of viable deletion mutants of polyoma virus. J Virol. 1979 Nov;32(2):523–529. doi: 10.1128/jvi.32.2.523-529.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Maness P. F., Engeser H., Greenberg M. E., O'Farrell M., Gall W. E., Edelman G. M. Characterization of the protein kinase activity of avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5028–5032. doi: 10.1073/pnas.76.10.5028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nathenson S. G., Uehara H., Ewenstein B. M., Kindt T. J., Coligan J. E. Primary structural: analysis of the transplantation antigens of the murine H-2 major histocompatibility complex. Annu Rev Biochem. 1981;50:1025–1052. doi: 10.1146/annurev.bi.50.070181.005113. [DOI] [PubMed] [Google Scholar]
  44. Novak U., Griffin B. E. Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus. Nucleic Acids Res. 1981 May 11;9(9):2055–2073. doi: 10.1093/nar/9.9.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Porter A. G., Barber C., Carey N. H., Hallewell R. A., Threlfall G., Emtage J. S. Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA. Nature. 1979 Nov 29;282(5738):471–477. doi: 10.1038/282471a0. [DOI] [PubMed] [Google Scholar]
  46. Powell J. T., Brew K. Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative. Biochemistry. 1976 Aug 10;15(16):3499–3505. doi: 10.1021/bi00661a016. [DOI] [PubMed] [Google Scholar]
  47. Robb R. J., Terhorst C., Strominger J. L. Sequence of the COOH-terminal hydrophilic region of histocompatibility antigens HLA-A2 and HLA-B7. J Biol Chem. 1978 Aug 10;253(15):5319–5324. [PubMed] [Google Scholar]
  48. Rose J. K., Welch W. J., Sefton B. M., Esch F. S., Ling N. C. Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3884–3888. doi: 10.1073/pnas.77.7.3884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schaffhausen B. S., Benjamin T. L. Phosphorylation of polyoma T antigens. Cell. 1979 Dec;18(4):935–946. doi: 10.1016/0092-8674(79)90206-x. [DOI] [PubMed] [Google Scholar]
  50. Schaffhausen B. S., Silver J. E., Benjamin T. L. Tumor antigen(s) in cell productively infected by wild-type polyoma virus and mutant NG-18. Proc Natl Acad Sci U S A. 1978 Jan;75(1):79–83. doi: 10.1073/pnas.75.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schaffhausen B., Benjamin T. L. Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro. J Virol. 1981 Oct;40(1):184–196. doi: 10.1128/jvi.40.1.184-196.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schlegel R., Benjamin T. L. Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities. Cell. 1978 Jul;14(3):587–599. doi: 10.1016/0092-8674(78)90244-1. [DOI] [PubMed] [Google Scholar]
  53. Sefton B. M., Hunter T., Beemon K. Temperature-sensitive transformation by Rous sarcoma virus and temperature-sensitive protein kinase activity. J Virol. 1980 Jan;33(1):220–229. doi: 10.1128/jvi.33.1.220-229.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Silver J., Schaffhausen B., Benjamin T. Tumor antigens induced by nontransforming mutants of polyoma virus. Cell. 1978 Oct;15(2):485–496. doi: 10.1016/0092-8674(78)90018-1. [DOI] [PubMed] [Google Scholar]
  55. Smith A. E., Smith R., Griffin B., Fried M. Protein kinase activity associated with polyoma virus middle T antigen in vitro. Cell. 1979 Dec;18(4):915–924. doi: 10.1016/0092-8674(79)90204-6. [DOI] [PubMed] [Google Scholar]
  56. Sottocasa G. L., Kuylenstierna B., Ernster L., Bergstrand A. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol. 1967 Feb;32(2):415–438. doi: 10.1083/jcb.32.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Staneloni R. J., Fluck M. M., Benjamin T. L. Host range selection of transformation-defective hr-t mutants of polyoma virus. Virology. 1977 Apr;77(2):598–609. doi: 10.1016/0042-6822(77)90485-8. [DOI] [PubMed] [Google Scholar]
  58. Tomita M., Marchesi V. T. Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2964–2968. doi: 10.1073/pnas.72.8.2964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Touster O., Aronson N. N., Jr, Dulaney J. T., Hendrickson H. Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes. J Cell Biol. 1970 Dec;47(3):604–618. doi: 10.1083/jcb.47.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Treisman R., Novak U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. doi: 10.1038/292595a0. [DOI] [PubMed] [Google Scholar]
  61. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  62. WINOCOUR E. Purification of polyoma virus. Virology. 1963 Feb;19:158–168. doi: 10.1016/0042-6822(63)90005-9. [DOI] [PubMed] [Google Scholar]
  63. Westcott K. R., Olwin B. B., Storm D. R. Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate. J Biol Chem. 1980 Sep 25;255(18):8767–8771. [PubMed] [Google Scholar]
  64. Witte O. N., Dasgupta A., Baltimore D. Abelson murine leukaemia virus protein is phosphorylated in vitro to form phosphotyrosine. Nature. 1980 Feb 28;283(5750):826–831. doi: 10.1038/283826a0. [DOI] [PubMed] [Google Scholar]
  65. Yankner B. A., Shooter E. M. Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1269–1273. doi: 10.1073/pnas.76.3.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES