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J. Biol. Chem., Vol. 281, Issue 7, 3954-3963, February 17, 2006

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Activation of the Checkpoint Kinase Rad53 by the Phosphatidyl Inositol Kinase-like Kinase Mec1^*

From the Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510

Saccharomyces cerevisiae Rad53, the ortholog of mammalian Chk2, is an essential protein kinase in DNA damage and DNA replication checkpoint pathways. Consecutive phosphatidyl inositol kinase-like kinase (PIKK)-dependent and PIKK-independent steps in activation of Rad53 are key steps for controlling and transmitting diverse downstream responses to DNA damage. However, these activities have not been demonstrated in vitro in defined systems. Here, we have shown that enzymatically dephosphorylated purified Rad53 autoactivates in vitro through a phosphorylation-dependent mechanism. Kinetic analysis demonstrated that autophosphorylation results in a more than 9-fold increase in protein kinase activity. Autophosphorylation was Rad53 concentration-dependent, indicating that the reaction follows an intermolecular mechanism. DNA damage induced oligomerization of a subset of Rad53 molecules in vivo. At low concentrations of Rad53, preincubation of Rad53 with immune complexes containing the Mec1/Ddc2 complex can activate Rad53 kinase activity. Our findings showed that Mec1/Ddc2 complexes can directly activate Rad53 through a phosphorylation-dependent mechanism, and more generally, supported the hypothesis that PIKKs regulate Chk2 orthologs through phosphorylation. Moreover, this work has substantiated a model for PIKK-independent amplification of Rad53 activation (and by extension, activation of other Chk2 orthologs) mediated by inter-Rad53 phosphorylation.

Received for publication, July 11, 2005 , and in revised form, November 29, 2005.

^* This work was supported by U. S. Public Health Service Grant R01CA82257. 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.

¹ Supported by U. S Army Medical Research and Materiel Command (USAMRMC) Grant DAMD 17-03-1-0355.

² Present address: Dept. of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94107.

³ Supported by USAMRMC Predoctoral Training Program in Breast Cancer Research DAMD Grant 17-99-1-946.

⁴ To whom correspondence should be addressed: Dept. of Pathology, Yale University School of Medicine, New Haven, CT 06510. Tel: 203-785-4832; Fax: 203-785-7467; E-mail: DF.Stern{at}yale.edu.

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