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

Article metrics


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 optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


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.


  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).

  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).

  3. 3

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

  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).

  5. 5

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

  6. 6

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

  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).

  8. 8

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

  9. 9

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

  10. 10

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

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  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).

  19. 19

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

  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).

  21. 21

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

  22. 22

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

  23. 23

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

  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).

  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).

  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).

  27. 27

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

  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).

  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).

  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).

  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).

Download references


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

Correspondence to Mien-Chie Hung.

Rights and permissions

Reprints and Permissions

About this article

Further reading