Phosphorylation of FOXO3a on Ser-7 by p38 Promotes Its Nuclear Localization in Response to Doxorubicin*

  1. Eric W.-F. Lam,1
  1. From the Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom,
  2. the §Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom,
  3. the Department of Physics, Imperial College London, London SW7 2AZ, United Kingdom, and
  4. the Institució Catalana de Recerca i Estudis Avançats and Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona, Spain
  1. 1 To whom correspondence should be addressed: Dept. of Surgery and Cancer, MRC Cyclotron Bldg., Imperial College London, Hammersmith Hospital Campus, Du Cane Rd., London W12 0NN, UK. E-mail: eric.lam{at}imperial.ac.uk.

Background: FOXO3a is a forkhead transcription factor that mediates the effects of doxorubicin in cancer treatment.

Results: p38 regulates FOXO3a nuclear translocation and phosphorylates FOXO3a on Ser-7 upon doxorubicin treatment.

Conclusion: p38 phosphorylation of FOXO3a on Ser-7 contributes to its nuclear relocalization and activation in response to doxorubicin.

Significance: This study provides new information on FOXO3a regulation and the molecular mechanism of action of doxorubicin.

Abstract

FOXO3a is a forkhead transcription factor that regulates a multitude of important cellular processes, including proliferation, apoptosis, differentiation, and metabolism. Doxorubicin treatment of MCF-7 breast carcinoma cells results in FOXO3a nuclear relocation and the induction of the stress-activated kinase p38 MAPK. Here, we studied the potential regulation of FOXO3a by p38 in response to doxorubicin. Co-immunoprecipitation studies in MCF-7 cells demonstrated a direct interaction between p38 and FOXO3a. We also showed that p38 can bind and phosphorylate a recombinant FOXO3a directly in vitro. HPLC-coupled phosphopeptide mapping and mass spectrometric analyses identified serine 7 as a major site for p38 phosphorylation. Using a phosphorylated Ser-7 FOXO3a antibody, we demonstrated that FOXO3a is phosphorylated on Ser-7 in response to doxorubicin. Immunofluorescence staining studies showed that upon doxorubicin treatment, the wild-type FOXO3a relocalized to the nucleus, whereas the phosphorylation-defective FOXO3a (Ala-7) mutant remained largely in the cytoplasm. Treatment with SB202190 also inhibits the doxorubicin-induced FOXO3a Ser-7 phosphorylation and nuclear accumulation in MCF-7 cells. In addition, doxorubicin caused the nuclear translocation of FOXO3a in wild-type but not p38-depleted mouse fibroblasts. Together, our results suggest that p38 phosphorylation of FOXO3a on Ser-7 is essential for its nuclear relocalization in response to doxorubicin.

Footnotes

  • * This work was supported by funds from Cancer Research UK (to K. K. H. and E. W.-F. L.), the Breast Cancer Campaign (to E. W.-F. L.), the Engineering and Physical Sciences Research Council (to D. J. K.), and Imperial College Healthcare NHS Trust-BRC Funding (to C.-Y. K.).

  • Graphic This article contains supplemental Figs. S1–S10.

  • Received July 19, 2011.
  • Revision received November 18, 2011.
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