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J. Biol. Chem., Vol. 278, Issue 27, 24428-24437, July 4, 2003

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Phosphorylation of Human Rad9 Is Required for Genotoxin-activated Checkpoint Signaling^*

From the ^aDepartment of Biochemistry and Molecular Biology, the ^gDepartment of Molecular Pharmacology and Experimental Therapeutics, the ^dProgram in Tumor Biology, the Divisions of ^eDevelopmental Oncology Research and ⁱRadiation Oncology, and the ^fBiomedical Mass Spectrometry and Functional Proteomics Facility, Mayo Clinic and Foundation, Rochester, Minnesota 55905 and the ^cCenter for Radiological Research, Columbia University, New York, New York 10032

Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in genotoxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9^–/– mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HU- and UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9^–/– cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.

Received for publication, February 13, 2003 , and in revised form, March 27, 2003.

Note Added in Proof—The mouse ES cells bearing a mutation in Mrad9 and described in this report were originally created and characterized by Kevin M. Hopkins, Wojtek Auerbach, Xiang Yuan Wang, M. Prakash Hande, Haiying Hang, Debra J. Wolgemuth, Alexandra L. Joyner, and Howard B. Lieberman. A more detailed characterization will appear in a separate paper.

^* This work was funded by National Institutes of Health Grants CA84321 (to L. M. K.), GM52493 (to H. B. L.), CA89816 (to H. B. L.), the Mayo Clinic Foundation (to L. M. K.), and the Magnus Ehrnrooth and Oskar Öflund Foundations (to P. R.-M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^b Present address: Turku Centre for Biotechnology Biocity, 5th floor, Tykistokatu 6B, Fin-20520 Turku, Finland.

^h Present address: Beyond Genomics, 40 Bear Hill Rd., Waltham, MA 02451.

^j To whom correspondence should be addressed: Division of Developmental Oncology Research, Guggenheim 13, Mayo Clinic and Foundation, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284-3124; Fax: 507-284-3906; E-mail: karnitz.larry{at}mayo.edu.

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