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J. Biol. Chem., Vol. 282, Issue 12, 9236-9243, March 23, 2007

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Identification of Carboxyl-terminal MCM3 Phosphorylation Sites Using Polyreactive Phosphospecific Antibodies^*

From the Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706

The functionally related ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases are critical regulators of DNA damage responses in mammalian cells. ATM and ATR share highly overlapping substrate specificities and show a strong preference for the phosphorylation of Ser or Thr residues followed by Gln. In this report we used a polyreactive phosphospecific antibody (-pDSQ) that recognizes a subset of phosphorylated Asp-Ser-Gln sequences to purify candidate ATM/ATR substrates. This led to the identification of phosphorylation sites in the carboxyl terminus of the minichromosome maintenance protein 3 (MCM3), a component of the hexameric MCM DNA helicase. We show that the -DSQ antibody recognizes tandem DSQ phosphorylation sites (Ser-725 and Ser-732) in the carboxyl terminus of murine MCM3 (mMCM3) and that ATM phosphorylates both sites in vitro. ATM phosphorylated the carboxyl termini of mMCM3 and human MCM3 in vivo and the phosphorylated form of MCM3 retained association with the canonical MCM complex. Although DNA damage did not affect steady-state levels of chromatin-bound MCM3, the ATM-phosphorylated form of MCM3 was preferentially localized to the soluble, nucleoplasmic fraction. This finding suggests that the carboxyl terminus of chromatin-loaded MCM3 may be sequestered from ATM-dependent checkpoint signals. Finally, we show that ATM and ATR jointly contribute to UV light-induced MCM3 phosphorylation, but that ATM is the predominant UV-activated MCM3 kinase in vivo. The carboxyl-terminal ATM phosphorylation sites are conserved in vertebrate MCM3 orthologs suggesting that this motif may serve important regulatory functions in response to DNA damage. Our findings also suggest that DSQ motifs are common phosphoacceptor motifs for ATM family kinases.

Received for publication, September 29, 2006 , and in revised form, January 22, 2007.

^* This work was supported by National Institutes of Health Grant GM067868-02, a Shaw Scientist Award (to R. S. T.) from the Greater Milwaukee Foundation, and a Post-doctoral Research Fellowship (to Y. S.) from The American Heart Association. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: 1300 University Ave., Madison, WI 53706. Tel.: 608-262-0027; E-mail: rstibbetts{at}wisc.edu.

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