Structural and biochemical insights into the regulation of protein phosphatase 2A by small t antigen of SV40

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Abstract

The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3–6, which overlaps with the binding site for the B′ (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B′ for the PP2A core enzyme. Consequently, ST does not efficiently displace B′ from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.

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Figure 1: Structure of the scaffolding subunit of PP2A bound to SV40 ST.
Figure 2: Structural features of the core domain of ST.
Figure 3: Recognition of the A subunit of PP2A by ST.
Figure 4: The J domain of ST directly contributes to binding of the PP2A core enzyme.
Figure 5: ST inhibits the phosphatase activity of the PP2A core enzyme.
Figure 6: Binding of ST and of B′ to the PP2A core enzyme are mutually exclusive.
Figure 7: A proposed mechanistic model of ST-mediated inhibition of PP2A.

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References

  1. 1

    Janssens, V. & Goris, J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439 (2001).

  2. 2

    Virshup, D.M. Protein phosphatase 2A: a panoply of enzymes. Curr. Opin. Cell Biol. 12, 180–185 (2000).

  3. 3

    Lechward, K., Awotunde, O.S., Swiatek, W. & Muszynska, G. Protein phosphatase 2A: variety of forms and diversity of functions. Acta Biochim. Pol. 48, 921–933 (2001).

  4. 4

    Kremmer, E., Ohst, K., Kiefer, J., Brewis, N. & Walter, G. Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells. Mol. Cell. Biol. 17, 1692–1701 (1997).

  5. 5

    Hemmings, B.A. et al. alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry 29, 3166–3173 (1990).

  6. 6

    Stone, S.R., Hofsteenge, J. & Hemmings, B.A. Molecular cloning of cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A. Biochemistry 26, 7215–7220 (1987).

  7. 7

    Green, D.D., Yang, S.I. & Mumby, M.C. Molecular cloning and sequence analysis of the catalytic subunit of bovine type 2A protein phosphatase. Proc. Natl. Acad. Sci. USA 84, 4880–4884 (1987).

  8. 8

    Arino, J., Woon, C.W., Brautigan, D.L., Miller, T.B., Jr . & Johnson, G.L. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes. Proc. Natl. Acad. Sci. USA 85, 4252–4256 (1988).

  9. 9

    Moreno, C.S. et al. WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A. J. Biol. Chem. 275, 5257–5263 (2000).

  10. 10

    Walter, G. & Mumby, M. Protein serine/threonine phosphatases and cell transformation. Biochim. Biophys. Acta 1155, 207–226 (1993).

  11. 11

    Janssens, V., Goris, J. & Van Hoof, C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34–41 (2005).

  12. 12

    Pallas, D.C. et al. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell 60, 167–176 (1990).

  13. 13

    Walter, G., Ruediger, R., Slaughter, C. & Mumby, M. Association of protein phosphatase 2A with polyoma virus medium tumor antigen. Proc. Natl. Acad. Sci. USA 87, 2521–2525 (1990).

  14. 14

    Sontag, E. et al. The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation. Cell 75, 887–897 (1993).

  15. 15

    Hahn, W.C. et al. Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol. Cell. Biol. 22, 2111–2123 (2002).

  16. 16

    Ruediger, R. et al. Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus. Mol. Cell. Biol. 12, 4872–4882 (1992).

  17. 17

    Ruediger, R., Hentz, M., Fait, J., Mumby, M. & Walter, G. Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens. J. Virol. 68, 123–129 (1994).

  18. 18

    Ruediger, R., Fields, K. & Walter, G. Binding specificity of protein phosphatase 2A core enzyme for regulatory B subunits and T antigens. J. Virol. 73, 839–842 (1999).

  19. 19

    Mateer, S.C., Fedorov, S.A. & Mumby, M.C. Identification of structural elements involved in the interaction of simian virus 40 small tumor antigen with protein phosphatase 2A. J. Biol. Chem. 273, 35339–35346 (1998).

  20. 20

    Scheidtmann, K.H., Mumby, M.C., Rundell, K. & Walter, G. Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen. Mol. Cell. Biol. 11, 1996–2003 (1991).

  21. 21

    Yang, S.I. et al. Control of protein phosphatase 2A by simian virus 40 small-t antigen. Mol. Cell. Biol. 11, 1988–1995 (1991).

  22. 22

    Kamibayashi, C. et al. Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. J. Biol. Chem. 269, 20139–20148 (1994).

  23. 23

    Cayla, X., Ballmer-Hofer, K., Merlevede, W. & Goris, J. Phosphatase 2A associated with polyomavirus small-T or middle-T antigen is an okadaic acid-sensitive tyrosyl phosphatase. Eur. J. Biochem. 214, 281–286 (1993).

  24. 24

    Van Hoof, C. & Goris, J. PP2A fulfills its promises as tumor suppressor: which subunits are important? Cancer Cell 5, 105–106 (2004).

  25. 25

    Pallas, D.C. et al. The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens. J. Virol. 66, 886–893 (1992).

  26. 26

    Chen, W. et al. Identification of specific PP2A complexes involved in human cell transformation. Cancer Cell 5, 127–136 (2004).

  27. 27

    Xing, Y. et al. Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, 341–352 (2006).

  28. 28

    Xu, Y. et al. Structure of the protein phosphatase 2A holoenzyme. Cell 127, 1239–1251 (2006).

  29. 29

    Cho, U.S. & Xu, W. Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445, 53–57 (2006).

  30. 30

    Groves, M.R., Hanlon, N., Turowski, P., Hemmings, B.A. & Barford, D. The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96, 99–110 (1999).

  31. 31

    Turk, B., Porras, A., Mumby, M.C. & Rundell, K. Simian virus 40 small-t antigen binds two zinc ions. J. Virol. 67, 3671–3673 (1993).

  32. 32

    Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).

  33. 33

    Jones, T.A., Zou, J.-Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

  34. 34

    Mungre, S. et al. Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation. J. Virol. 68, 1675–1681 (1994).

  35. 35

    Campbell, K.S., Auger, K.R., Hemmings, B.A., Roberts, T.M. & Pallas, D.C. Identification of regions in polyomavirus middle T and small t antigens important for association with protein phosphatase 2A. J. Virol. 69, 3721–3728 (1995).

  36. 36

    Kim, H.Y., Ahn, B.Y. & Cho, Y. Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen. EMBO J. 20, 295–304 (2001).

  37. 37

    Cayla, X. et al. Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes. Biochemistry 29, 658–667 (1990).

  38. 38

    Van Hoof, C., Cayla, X., Bosch, M., Merlevede, W. & Goris, J. The phosphotyrosyl phosphatase activator of protein phosphatase 2A. A novel purification method, immunological and enzymic characterization. Eur. J. Biochem. 226, 899–907 (1994).

  39. 39

    Srinivasan, A. et al. The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain. Mol. Cell. Biol. 17, 4761–4773 (1997).

  40. 40

    Kamibayashi, C., Lickteig, R.L., Estes, R., Walter, G. & Mumby, M.C. Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits. J. Biol. Chem. 267, 21864–21872 (1992).

  41. 41

    Chao, Y. et al. Structure and mechanism of the phosphotyrosyl phosphatase activator. Mol. Cell 23, 535–546 (2006).

  42. 42

    Johnson, S.A. & Hunter, T. Kinomics: methods for deciphering the kinome. Nat. Methods 2, 17–25 (2005).

  43. 43

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  44. 44

    McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D. Biol. Crystallogr. 61, 458–464 (2005).

  45. 45

    Brunger, A.T. et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

  46. 46

    Kraulis, P.J. Molscript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst. 24, 946–950 (1991).

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Acknowledgements

We thank T. Roberts at Harvard Medical School for the complementary DNA encoding SV40 ST, and A. Saxena at the beamlines of the National Synchrotron Light Source, Brookhaven National Laboratory for help. This work was supported by grant R01-CA123155 from the US National Institutes of Health (Y.S.).

Author information

Y.C. and Y. Xu designed, performed and analyzed most of the experiments. Q.B. contributed to PP2A enzymology. Y. Xing, Z. Li and Z. Lin provided technical assistance. J.B.S. contributed to discussions. P.D.J. refined the structure. Y.S. led the team and wrote the paper.

Correspondence to Yigong Shi.

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