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

• Published in: The Journal of biological chemistry Vol. 287; no. 2; pp. 1545 - 1555
• Main Authors: Ho, Ka-Kei, McGuire, Victoria A., Koo, Chuay-Yeng, Muir, Kyle W., de Olano, Natalia, Maifoshie, Evie, Kelly, Douglas J., McGovern, Ursula B., Monteiro, Lara J., Gomes, Ana R., Nebreda, Angel R., Campbell, David G., Arthur, J. Simon C., Lam, Eric W.-F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.01.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Active Transport, Cell Nucleus - drug effects; Active Transport, Cell Nucleus - genetics; Amino Acid Substitution; Animals; Antibiotics, Antineoplastic - pharmacology; Breast Cancer; Cancer Therapy; Cell Line, Tumor; Cell Nucleus - genetics; Cell Nucleus - metabolism; Doxorubicin - pharmacology; Enzyme Inhibitors - pharmacology; Forkhead Box Protein O3; Forkhead Transcription Factors - genetics; Forkhead Transcription Factors - metabolism; HEK293 Cells; Humans; Imidazoles - pharmacology; Mice; Mice, Knockout; Molecular Bases of Disease; Mutation, Missense; Nuclear Translocation; p38 MAPK; p38 Mitogen-Activated Protein Kinases - antagonists & inhibitors; p38 Mitogen-Activated Protein Kinases - genetics; p38 Mitogen-Activated Protein Kinases - metabolism; Phosphorylation - drug effects; Phosphorylation - genetics; Pyridines - pharmacology; Signal Transduction; Transcription Factors; Cancer Therapy; Transcription Factors; Signal Transduction; Nuclear Translocation; Breast Cancer; p38 MAPK
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.284224

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. 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.