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Molecular and Cellular Biology, July 2001, p. 4129-4139, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4129-4139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1

Hui Zhao1 and Helen Piwnica-Worms1,2,*

Department of Cell Biology and Physiology1 and Howard Hughes Medical Institute,2 Washington University School of Medicine, St. Louis, Missouri 63110-1093

Received 7 February 2001/Returned for modification 19 March 2001/Accepted 23 March 2001

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.

* Corresponding author. Mailing address: Department of Cell Biology and Physiology and Howard Hughes Medical Institute, Washington University School of Medicine, Box 8228, 660 South Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-6812. Fax: (314) 362-3709. E-mail: hpiwnica{at}cellbio.wustl.edu.

Molecular and Cellular Biology, July 2001, p. 4129-4139, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4129-4139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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