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The nuclear import function of Smad2 is masked by SARA and unmasked by TGFb-dependent phosphorylation

Nature Cell Biology volume 2, pages 559–562 (2000)Cite this article

Nuclear accumulation of Smad transcription factors in response to transforming growth factor β CTGFβ cytokines is triggered by TGFβ-receptor-mediated phosphorylation1. Receptor phosphorylation of Smad2, which occurs in the extreme carboxy-terminal sequence SSMS, allows association of this protein with the related protein Smad4. Smad4 is important for assembly of transcriptional complexes, but is not required for nuclear accumulation2. The mechanism by which receptor-mediated phosphorylation promotes nuclear accumulation of Smad proteins has thus remained unknown. Here we show that Smad2 has intrinsic nuclear import activity that does not require TGFβ receptor-mediated phosphorylation. The Mad-homology domain 2 (MH2) of Smad2 drives nuclear import by a selective mechanism that is distinct from the classical mechanism for nuclear import that involves a cluster of basic residues known as the nuclear localization signal (NLS)3,4,5. Nuclear import of unphosphorylated Smad2 is inhibited by contact with the Smad-binding domain of the Smad anchor for receptor activation (SARA), a protein that was previously shown to mediate access of Smad to the TGFβ receptor6. Receptor-mediated phosphorylation reduces the affinity of Smad2 for SARA, releasing Smad2 and unmasking its intrinsic nuclear-import activity.

To investigate Smad2 nuclear import, we used an in vitro system that was previously used to delineate the NLS nuclear import pathway. This system uses digitonin to selectively permeabilize the plasma membrane in HeLa cells, leaving the nuclear membrane as an intact barrier7,8. Cytosolic factors are largely depleted by diffusion so that, to enter the nucleus under these conditions, NLS-containing proteins require cytosolic factors that include importin-α, which recognizes the NLS, and importin-β, which brings the complex to the nuclear pore7. We validated the suitability of HeLa cells by showing that TGFβ induces rapid nuclear accumulation of endogenous Smad2 in this cell line (see Supplementary Information , Fig. S1).

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Figure 1: The MH2 domain mediates nuclear import of Smad2 by a distinct mechanism.
Figure 2: Full-length Smad2 is imported into the nucleus without phosphorylation of its SSMS motif.
Figure 3: SARA interaction prevents nuclear import of Smad2, and phosphorylation relieves Smad2 from SARA, allowing its entry into the nucleus.

References

  1. Massagué, J. Annu. Rev. Biochem. 67, 753–791 (1998).

  2. Liu, F., Pouponnot, C. & Massagué, J. Genes Dev. 11, 3157–3167 (1997).

    Article  CAS  Google Scholar 

  3. Rexach, M. & Blobel, G. Cell 83, 683–692 (1995).

    Article  CAS  Google Scholar 

  4. Mattaj, I. W. & Englmeier, L. Annu. Rev. Biochem. 67, 265–306 (1998).

    Article  CAS  Google Scholar 

  5. Nakielny, S. & Dreyfuss, G. Cell 99, 677–690 (1999).

    Article  CAS  Google Scholar 

  6. Tsukazaki, T., Chiang, T. A., Davison, A. F., Attisano, L. & Wrana, J. L. Cell 95, 779–791 (1998).

    Article  CAS  Google Scholar 

  7. Adam, S. A., Marr, R. S. & Gerace, L. J. Cell Biol. 111, 807–816 (1990).

    Article  CAS  Google Scholar 

  8. Adam, S. A., Sterne-Marr, R. & Gerace, L. Methods Enzymol. 219, 97–110 (1992).

    Article  CAS  Google Scholar 

  9. Massagué, J. & Chen, Y. G. Genes Dev. 14, 627–644 (2000).

    PubMed  Google Scholar 

  10. Pollard, V. W. et al. Cell 86, 985–994 (1996).

    Article  CAS  Google Scholar 

  11. Baker, J. & Harland, R. M. Genes Dev. 10, 1880–1889 (1996).

    Article  CAS  Google Scholar 

  12. Shi, Y. et al. Cell 94, 585–594 (1998).

    Article  CAS  Google Scholar 

  13. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. Cell 94, 193–204 (1998).

    Article  CAS  Google Scholar 

  14. Gorlich, D., Henklein, P., Laskey, R. A. & Hartmann, E. EMBO J. 15, 1810–1817 (1996).

    Article  CAS  Google Scholar 

  15. Macias-Silva, M. et al. Cell 87, 1215–1224 (1996).

    Article  CAS  Google Scholar 

  16. Kretzschmar, M., Liu, F., Hata, A., Doody, J. & Massagué, J. Genes Dev. 11, 984–995 (1997).

    Article  CAS  Google Scholar 

  17. Wu, G. et al. Science 287, 92–97 (2000).

    Article  CAS  Google Scholar 

  18. Dong, C., Li, Z., Alvarez, R., Feng, X-H. & Goldschmidt-Clermont, P. J. Mol. Cell 5, 27–34 (2000).

    Article  CAS  Google Scholar 

  19. Kretzschmar, M., Doody, J., Timokhina, I. & Massagué, J. Genes Dev. 13, 804–816 (1999).

    Article  CAS  Google Scholar 

  20. Wieser, R., Wrana, J. L. & Massagué, J. EMBO J. 14, 2199–2208 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Blobel and B. Gumbiner for suggestions on nuclear import analysis, D. Görlich for plasmids encoding IBB and importin-β, J. Wrana for expression plasmids for GST–SBD and Myc–SARA, Y-G. Shi for Smad2-encoding baculovirus, and E-K. Suh and B. Gumbiner for NLS–HA and for technical advice concerning the nuclear-import assay. This work was supported by NIH grant CA34610. L.X. is supported by a Damon Runyon-Walter Winchell Fellowship (DRG–1540) of the Cancer Research Fund, and J.M. is an Investigator of the Howard Hughes Medical Institute.

Correspondence and requests for materials should be addressed to J.M. Supplementary Information is available on Nature Cell Biology’s World-Wide Web site (http://www.nature.com/ncb) or as paper copy from the London editorial office of Nature Cell Biology.

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  1. Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Lan Xu, Ye-Guang Chen & Joan Massagué

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Supplementary information

Figure 1

TGFβ induced nuclear accumulation of Smad2/3 in HeLa cells. (PDF 310 kb)

Figure 2 Left panel. Coomassie Blue staining of purified full length Smad2.

Figure 3 Fusion of GFP to the N-terminus of MH2 domain disrupts its interaction with SARA.

Figure 4 Preparation of phosphorylated Smad2.

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Xu, L., Chen, YG. & Massagué, J. The nuclear import function of Smad2 is masked by SARA and unmasked by TGFb-dependent phosphorylation. Nat Cell Biol 2, 559–562 (2000). https://doi.org/10.1038/35019649

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