Phosphorylation of FOXO3a on Ser-7 by p38 promotes its nuclear localization in response to doxorubicin

Ka-Kei Ho, Victoria A. McGuire, Chuay-Yeng Koo, Kyle W. Muir, Natalia de Olano, Evie Maifoshie, Douglas J. Kelly, Ursula B. McGovern, Lara J. Monteiro, Ana R. Gomes, Angel R. Nebreda, David G. Campbell, J. Simon C. Arthur, Eric W. -F. Lam

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    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.

    Original languageEnglish
    Pages (from-to)1545-1555
    Number of pages11
    JournalJournal of Biological Chemistry
    Volume287
    Issue number2
    DOIs
    Publication statusPublished - 6 Jan 2012

    Cite this

    Ho, K-K., McGuire, V. A., Koo, C-Y., Muir, K. W., de Olano, N., Maifoshie, E., Kelly, D. J., McGovern, U. B., Monteiro, L. J., Gomes, A. R., Nebreda, A. R., Campbell, D. G., Arthur, J. S. C., & Lam, E. W. -F. (2012). Phosphorylation of FOXO3a on Ser-7 by p38 promotes its nuclear localization in response to doxorubicin. Journal of Biological Chemistry, 287(2), 1545-1555. https://doi.org/10.1074/jbc.M111.284224