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

Abstract

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.

ACKNOWLEDGMENTS

We are grateful to J. Schwarz and C. Ryan for assistance with production of recombinant adenoviruses and J. Gales for production and purification of antibodies. We thank K. Cimprich and R. Abraham for providing ATR reagents and M. Linder for helpful discussions. We thank J. Hurov, C. Lovly, and M.-S. Chen for comments on the manuscript.

This work was supported by a grant from the National Institutes of Health. H.P.-W. is an Investigator of the Howard Hughes Medical Institute.