Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells

Abstract

Amplification or overexpression of HER-2/neu in cancer cells confers resistance to apoptosis and promotes cell growth. The cellular localization of p21Cip1/WAF1 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Here we show that HER-2/neu-mediated cell growth requires the activation of Akt, which associates with p21Cip1/WAF1 and phosphorylates it at threonine 145, resulting in cytoplasmic localization of p21Cip1/WAF1. Furthermore, blocking the Akt pathway with a dominant-negative Akt mutant restores the nuclear localization and cell-growth-inhibiting activity of p21Cip1/WAF1. Our results indicate that HER-2/neu induces cytoplasmic localization of p21Cip1/WAF1 through activation of Akt to promote cell growth, which may have implications for the oncogenic activity of HER-2/neu and Akt.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The Akt pathway is required for HER-2/neu-mediated cell proliferation.
Figure 2: Threonine 145 of p21Cip1/WAF1 is phosphorylated in vivo.
Figure 3: Akt interacts with p21Cip1/WAF1 and phosphorylates it at threonine 145.
Figure 4: Akt alters the cellular localization of p21Cip1/WAF1.
Figure 5: Cellular localization of endogenous p21Cip1/WAF1.
Figure 6: HER-2/neu activates Akt and induces cytoplasmic localization of p21Cip1/WAF1 in breast tumours.
Figure 7: Growth-inhibiting activities of p21Cip1/WAF1 mutants.
Figure 8: Model for HER-2/neu-induced cytoplasmic localization of p21Cip1/WAF1 through activation of Akt.

References

  1. 1

    Slamon, D. J. et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177–182 (1987).

    CAS  Article  Google Scholar 

  2. 2

    Slamon, D. J. et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707–712 (1989).

    CAS  Article  Google Scholar 

  3. 3

    Yu, D. & Hung, M-C. in DNA Alterations in Cancer (ed. Ehrlich, M.) Ch. 21 (Natick, Massachusetts, 2000).

    Google Scholar 

  4. 4

    Zhou, B. P. et al. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kB pathways. J. Biol. Chem. 275, 8027–8031 (2000).

    CAS  Article  Google Scholar 

  5. 5

    Downward, J. Mechanisms and consequences of activation of protein kinase B/Akt. Curr. Opin. Cell Biol. 10, 262–267 (1998).

    CAS  Article  Google Scholar 

  6. 6

    Datta, S. R., Brunet, A. & Greenberg, M. E. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927 (1999).

    CAS  Article  Google Scholar 

  7. 7

    Peso, L. D., Gonzalez-Garcia, M., Page, C., Herrera, R. & Nunez, G. Interleukin-3-induced phosphorylation of bad through the protein kinase Akt. Science 278, 687–689 (1997).

    Article  Google Scholar 

  8. 8

    Cardone, M. H. et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318–1321 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Brunet, A. et al. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96, 857–868 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Kops., G. J. et al. Direct control of the forkhead transcription factor AFX by protein kinase B. Nature 398, 630–634 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Ozes, O. N. et al. NF-kB activation by tumor necrosis factor requires the Akt serine-threonine kinase. Nature 401, 82–85 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Romashkova, J. A. & Makarov, S. S. NF-kB is a target of Akt in anti-apoptotic PDGF signalling. Nature 401, 86–90 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Ahmed, N. N., Gries, H. L., Bellacosa, A., Chan, T. O. & Tsichlis, P. N. Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc. Natl Acad. Sci. USA 94, 3627–3632 (1997).

    CAS  Article  Google Scholar 

  14. 14

    Brennan, P. et al. Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F. Immunity 7, 679–689 (1997).

    CAS  Article  Google Scholar 

  15. 15

    Medema, R. H., Kops, G. J., Bos, J. L. & Burgering, B. M. T. AFX-like forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27Kip1. Nature 404, 782–787 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Andjelkovic, M. et al. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272, 31515–31524 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Meier, R., Alessi, D. R., Cron, P., Andjelkovic, M. & Hemmings, B. A. Mitogenic activation, phosphorylation and nuclear translocation of protein kinase B. J. Biol. Chem. 272, 30491–30497 (1997).

    CAS  Article  Google Scholar 

  18. 18

    Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. The p21 Cdk-interacting protein Cip is a potent inhibitor of G1 cyclin-dependent kinase. Cell 75, 805–816 (1993).

    CAS  Article  Google Scholar 

  19. 19

    El-Deiry, W. S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993).

    CAS  Article  Google Scholar 

  20. 20

    Noda, A., Ning, Y., Venable, S. F., Pereira-Smith, O. M. & Smith, J. R. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res. 211, 90–98 (1994).

    CAS  Article  Google Scholar 

  21. 21

    Sherr, C. J. & Roberts, J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9, 1149–1163 (1995).

    CAS  Article  Google Scholar 

  22. 22

    Goubin, F. & Ducommun, B. Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. Oncogene 10, 2281–2287 (1995).

    CAS  PubMed  Google Scholar 

  23. 23

    Asada, M. et al. Apoptosis inhibitory activity of cytoplasmic p21Cip1/WAF1 in monocytic differentiation. EMBO J. 18, 1223–1234 (1999).

    CAS  Article  Google Scholar 

  24. 24

    Yu, D. et al. Overexpression of ErbB2 blocks taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol. Cell 2, 581–591 (1998).

    CAS  Article  Google Scholar 

  25. 25

    Scott, M. T., Morrice, N. & Ball, K. L. Reversible phosphorylation at the C-terminal regulatory domain of p21Waf1/Cip1 modulates proliferating cell nuclear antigen binding. J. Biol. Chem. 275, 11529–11537 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Chen, J., Jackson, P. K., Kirschner, M. W. & Dutta, A. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature 374, 386–388 (1995).

    CAS  Article  Google Scholar 

  27. 27

    Porter, A. G. Protein translocation in apoptosis. Trends Cell Biol. 9, 394–401 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Diehl, J. A., Cheng, M., Roussel, M. F. & Sherr, C. J. Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 12, 3499 (1998).

    CAS  Article  Google Scholar 

  29. 29

    Shao, R. et al. Inhibition of nuclear factor-kB activity is involved in E1A-mediated sensitization of radiation-induced apoptosis. J. Biol. Chem. 272, 32739–32742 (1997).

    CAS  Article  Google Scholar 

  30. 30

    Van der Geer, P. & Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates. Electrophoresis 15, 544–554 (1994).

    CAS  Article  Google Scholar 

  31. 31

    Gatti, A. & Traugh, T. A. A two-dimensional peptide gel electrophoresis system for phosphopeptide mapping and amino acid sequencing. Anal. Biochem. 266, 198–204 (1999).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank D. Yu, R. B. Arlinghaus and W. H. Klein for critical comments on the manuscript. This work was supported by grants from the National Institutes of Health (to M-C.H.), by a SPORE grant in ovarian cancer (to M-C.H.), and by the Nellie Connally Breast Cancer Research Fund at the M. D. Anderson Cancer Center (M-C.H.). B.P.Z. is a recipient of a postdoctoral fellowship from the US Department of Defense Breast Cancer Research.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Mien-Chie Hung.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhou, B., Liao, Y., Xia, W. et al. Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol 3, 245–252 (2001). https://doi.org/10.1038/35060032

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing