Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance

Nature Cell Biology volume 7, pages 591–600 (2005)Cite this article

Abstract

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Human kinase siRNA library screen.
Figure 2: Human phosphatase siRNA library screen.
Figure 3: Human phosphatase siRNA library screen identifies cell death phosphatases and resistance to apoptosis.
Figure 4: Low-dose Taxol and siRNAs directed against survival kinases lead to enhanced apoptosis.
Figure 5: Synergistic chemotherapy-induced apoptosis in BT474 breast carcinoma cells with survival kinase siRNAs.

Similar content being viewed by others

References

  1. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57–70 (2000).

    Article  CAS  Google Scholar 

  2. MacKeigan, J.P., Collins, T.S. & Ting, J.P. MEK inhibition enhances paclitaxel-induced tumor apoptosis. J. Biol. Chem. 275, 38953–38956 (2000).

    Article  CAS  Google Scholar 

  3. Slamon, D.J. et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med. 344, 783–792 (2001).

    Article  CAS  Google Scholar 

  4. MacKeigan, J.P. et al. Inactivation of the antiapoptotic phosphatidylinositol 3-kinase-Akt pathway by the combined treatment of taxol and mitogen-activated protein kinase kinase inhibition. Clin. Cancer Res. 8, 2091–2099 (2002).

    CAS  PubMed  Google Scholar 

  5. Pietras, K. et al. Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res. 62, 5476–5484 (2002).

    CAS  PubMed  Google Scholar 

  6. Hannon, G.J. & Rossi, J.J. Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378 (2004).

    Article  CAS  Google Scholar 

  7. Tassi, E., Biesova, Z., Di Fiore, P.P., Gutkind, J.S. & Wong, W.T. Human JIK, a novel member of the STE20 kinase family that inhibits JNK and is negatively regulated by epidermal growth factor. J. Biol. Chem. 274, 33287–33295 (1999).

    Article  CAS  Google Scholar 

  8. Wishart, M.J., Denu, J.M., Williams, J.A. & Dixon, J.E. A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase. J. Biol. Chem. 270, 26782–26785 (1995).

    Article  CAS  Google Scholar 

  9. Wishart, M.J. & Dixon, J.E. Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem. Sci. 23, 301–306 (1998).

    Article  CAS  Google Scholar 

  10. Schiff, P.B., Fant, J. & Horwitz, S.B. Promotion of microtubule assembly in vitro by taxol. Nature 277, 665–667 (1979).

    Article  CAS  Google Scholar 

  11. Schiff, P.B. & Horwitz, S.B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl Acad. Sci. USA 77, 1561–1565 (1980).

    Article  CAS  Google Scholar 

  12. Sorenson, C.M., Barry, M.A. & Eastman, A. Analysis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin. J. Natl Cancer Inst. 82, 749–755 (1990).

    Article  CAS  Google Scholar 

  13. van Maanen, J.M., Retel, J., de Vries, J. & Pinedo, H.M. Mechanism of action of antitumor drug etoposide: a review. J. Natl Cancer Inst. 80, 1526–1533 (1988).

    Article  CAS  Google Scholar 

  14. Brunet, A. et al. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol. Cell. Biol. 21, 952–965 (2001).

    Article  CAS  Google Scholar 

  15. Huang, S. & Houghton, P.J. Inhibitors of mammalian target of rapamycin as novel antitumor agents: from bench to clinic. Curr. Opin. Investig. Drugs 3, 295–304 (2002).

    CAS  PubMed  Google Scholar 

  16. Bjornsti, M.A. & Houghton, P.J. The TOR pathway: a target for cancer therapy. Nature Rev. Cancer 4, 335–348 (2004).

    Article  CAS  Google Scholar 

  17. Fingar, D.C. & Blenis, J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23, 3151–3171 (2004).

    Article  CAS  Google Scholar 

  18. Senderowicz, A.M. Small-molecule cyclin-dependent kinase modulators. Oncogene 22, 6609–6620 (2003).

    Article  CAS  Google Scholar 

  19. Pasleau, F., Grooteclaes, M. & Gol-Winkler, R. Expression of the c-erbB2 gene in the BT474 human mammary tumor cell line: measurement of c-erbB2 mRNA half-life. Oncogene 8, 849–854 (1993).

    CAS  PubMed  Google Scholar 

  20. Lane, H.A. et al. ErbB2 potentiates breast tumor proliferation through modulation of p27Kip1-Cdk2 complex formation: receptor overexpression does not determine growth dependency. Mol. Cell. Biol. 20, 3210–3223 (2000).

    Article  CAS  Google Scholar 

  21. Bonifati, V. et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science 299, 256–259 (2003).

    Article  CAS  Google Scholar 

  22. Valente, E.M. et al. Hereditary early-onset Parkinson's disease caused by mutations in PINK1. Science 304, 1158–1160 (2004).

    Article  CAS  Google Scholar 

  23. MacKeigan, J.P. et al. Proteomic profiling drug-induced apoptosis in non-small cell lung carcinoma: identification of RS/DJ-1 and RhoGDIα. Cancer Res. 63, 6928–6934 (2003).

    CAS  PubMed  Google Scholar 

  24. Cheng, J.Q. et al. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc. Natl Acad. Sci. USA 89, 9267–9271 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Storz, P. & Toker, A. Protein kinase D mediates a stress-induced NF-κB activation and survival pathway. EMBO J. 22, 109–120 (2003).

    Article  CAS  Google Scholar 

  27. Yang, B.F., Xiao, C., Roa, W.H., Krammer, P.H. & Hao, C. Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of Fas-mediated signaling in malignant glioma cells. J. Biol. Chem. 278, 7043–7050 (2003).

    Article  CAS  Google Scholar 

  28. Seton-Rogers, S.E. et al. Cooperation of the ErbB2 receptor and transforming growth factor β in induction of migration and invasion in mammary epithelial cells. Proc. Natl Acad. Sci. USA 101, 1257–1262 (2004).

    Article  CAS  Google Scholar 

  29. Muraoka, R.S. et al. Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factor β1. Mol. Cell. Biol. 23, 8691–8703 (2003).

    Article  CAS  Google Scholar 

  30. Siegel, P.M., Shu, W., Cardiff, R.D., Muller, W.J. & Massague, J. Transforming growth factor β signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis. Proc. Natl Acad. Sci. USA 100, 8430–8435 (2003).

    Article  CAS  Google Scholar 

  31. Di Cristofano, A., Pesce, B., Cordon-Cardo, C. & Pandolfi, P.P. Pten is essential for embryonic development and tumour suppression. Nature Genet. 19, 348–355 (1998).

    Article  CAS  Google Scholar 

  32. Goberdhan, D.C., Paricio, N., Goodman, E.C., Mlodzik, M. & Wilson, C. Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway. Genes Dev. 13, 3244–3258 (1999).

    Article  CAS  Google Scholar 

  33. Stambolic, V. et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29–39 (1998).

    Article  CAS  Google Scholar 

  34. Suzuki, A. et al. High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr. Biol. 8, 1169–1178 (1998).

    Article  CAS  Google Scholar 

  35. Murphy, L.O., MacKeigan, J.P. & Blenis, J. A network of immediate early gene products propagates subtle differences in mitogen-activated protein kinase signal amplitude and duration. Mol. Cell. Biol. 24, 144–153 (2004).

    Article  CAS  Google Scholar 

  36. Alvarez, B., Martinez, A.C., Burgering, B.M. & Carrera, A.C. Forkhead transcription factors contribute to execution of the mitotic programme in mammals. Nature 413, 744–747 (2001).

    Article  CAS  Google Scholar 

  37. Wang, Z. et al. Mutational analysis of the tyrosine phosphatome in colorectal cancers. Science 304, 1164–1166 (2004).

    Article  CAS  Google Scholar 

  38. Narayan, G. et al. Genetic analysis identifies putative tumor suppressor sites at 2q35-q36.1 and 2q36.3-q37.1 involved in cervical cancer progression. Oncogene 22, 3489–3499 (2003).

    Article  CAS  Google Scholar 

  39. Brondello, J.M., Pouyssegur, J. & McKenzie, F.R. Reduced MAP kinase phosphatase-1 degradation after p42/p44MAPK-dependent phosphorylation. Science 286, 2514–2517 (1999).

    Article  CAS  Google Scholar 

  40. Makin, G. & Dive, C. Modulating sensitivity to drug-induced apoptosis: the future for chemotherapy? Breast Cancer Res. 3, 150–153 (2001).

    Article  CAS  Google Scholar 

  41. Manning, G., Whyte, D.B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    Article  CAS  Google Scholar 

  42. Tartaglia, M. et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nature Genet. 29, 465–468 (2001).

    Article  CAS  Google Scholar 

  43. Mohi, M.G. et al. Prognostic, therapeutic, and mechanistic implications of a mouse model of leukemia evoked by Shp2 (PTPN11) mutations. Cancer Cell 7, 179–191 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge E. Lader and S. Yerramilli for their assistance with siRNA library design and QRT–PCR expertise. We thank F. Tsai for help with data analysis, W. Dowdle for technical assistance, and C. Dimitri, B. Ballif and members of the Blenis laboratory for their critical feedback. This work is supported by National Institutes of Health grants RO1CA46595 and GM51405 to J.B. J.M. is a Fellow of the American Cancer Society and L.M. is a Special Fellow of the Leukemia & Lymphoma Society.

Author information

Author notes

  1. Jeffrey P. MacKeigan, Leon O. Murphy and John Blenis: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Jeffrey P. MacKeigan, Leon O. Murphy & John Blenis

Authors
  1. Jeffrey P. MacKeigan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leon O. Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John Blenis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John Blenis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary figures S1, S2, S3 and S4 (PDF 124 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

MacKeigan, J., Murphy, L. & Blenis, J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat Cell Biol 7, 591–600 (2005). https://doi.org/10.1038/ncb1258

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1258

Search

Advanced 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