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FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway

Nature Structural & Molecular Biology volume 15, pages 1138–1146 (2008)Cite this article

Abstract

In response to DNA damage or replication fork stress, the Fanconi anemia pathway is activated, leading to monoubiquitination of FANCD2 and FANCI and their colocalization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in six conserved and clustered Ser/Thr-Gln motifs of FANCI largely abrogate monoubiquitination and focus formation of both FANCI and FANCD2, resulting in loss of DNA repair function. Conversely, FANCI carrying phosphomimic mutations on the same six residues induces constitutive monoubiquitination and focus formation of FANCI and FANCD2, and protects against cell killing and chromosome breakage by DNA interstrand cross-linking agents. We propose that the multiple phosphorylation of FANCI serves as a molecular switch in activation of the Fanconi anemia pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

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Figure 1: Gene targeting of FANCI resulted in loss of FANCD2 monoubiquitination and focus formation.
Figure 2: FANCI monoubiquitination has a minor role in inducing Fanconi anemia pathway activation.
Figure 3: FANCI monoubiquitination depends on FANCD2 monoubiquitination, but their physical interaction is constitutive.
Figure 4: Phosphorylation sites in FANCI are required for Fanconi anemia pathway activation and DNA repair.
Figure 5: Phosphomimic mutants induce constitutive activation of the Fanconi anemia pathway.
Figure 6: Analysis of phosphorylation mutants in human FANCI.

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Acknowledgements

We would like to thank T. Natsume (National Institute of Advanced Industrial Science and Technology, Japan), K. Komatsu (Kyoto University), H. Kurumizaka (Waseda University), J. Lin and J.W. Harper (Harvard Medical School) for reagents; R. Sasaki (Digital Microsystems) for advice and help in microscopy; K. Namikoshi for expert technical help; and H. Shimamoto and S. Arai for secretarial assistance. This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (M.T.), by grants from Ministry of Health, Labour, and Welfare (M.T.), and by US National Institute of Allergy and Infectious Diseases grant 1U19A1067751 and US National Institutes of Health grants (S.J.E.). A. Smogorzewska is supported by T32CA09216 to the MGH Pathology Department. Financial support was also provided by The Novartis Foundation (Japan) for the Promotion of Science (M.T.) and The Uehara Memorial Foundation (M.T.). S.J.E. is funded by the Howard Hughes Medical Institute.

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Author notes

  1. Hiroyuki Kitao & Alihossein Saberi

    Present address: Present addresses: Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan (H.K.); Department of Genetics, School of Medicine, Ahwaz Jondishapoor of Medical Sciences, Golestan Street, Ahwaz 61357-15749, Iran (A. Saberi).,

  2. Masamichi Ishiai and Hiroyuki Kitao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Late Effect Studies, Laboratory of DNA Damage Signaling, Radiation Biology Center, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, 606-8501, Kyoto, Japan

    Masamichi Ishiai, Hiroyuki Kitao, Junya Tomida, Emi Uchida, Alihossein Saberi & Minoru Takata

  2. Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, 734-8553, Hiroshima, Japan

    Hiroyuki Kitao, Aiko Kinomura & Minoru Takata

  3. Department of Genetics, Howard Hughes Medical Institute, Center for Genetics and Genomics, Brigham and Women's Hospital, Harvard Medical School, Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Agata Smogorzewska & Stephen J Elledge

  4. Department of Pathology, Massachusetts General Hospital, 55 Fruit Street, WRN 225, Boston, 02114, Massachusetts, USA

    Agata Smogorzewska

  5. Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, 734-8553, Hiroshima, Japan

    Eiji Kinoshita, Emiko Kinoshita-Kikuta & Tohru Koike

  6. Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, 734-8553, Hiroshima, Japan

    Satoshi Tashiro

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  1. Masamichi Ishiai
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Contributions

M.T., A. Smogorzewska and S.J.E. conceived and designed the experiments; M.I., H.K., A. Saberi and E.U. performed most of the DT40 cell experiments; M.I., A.K. and S.T. performed microscopic analysis; E.K., E.K.-K., T.K. and J.T. designed and performed Phos-tag experiments; A. Smogorzewska performed human cell experiments; M.I. and H.K. analyzed the DT40 cell data; M.T., A. Smogorzewska and S.J.E. wrote the paper.

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Correspondence to Minoru Takata.

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Ishiai, M., Kitao, H., Smogorzewska, A. et al. FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway. Nat Struct Mol Biol 15, 1138–1146 (2008). https://doi.org/10.1038/nsmb.1504

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