ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.
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.
Zhao
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Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Piwnica-Worms
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D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000970:Antineoplastic Agents / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D018797:Cell Cycle Proteins; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002945:Cisplatin / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D004249:DNA Damage; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D015151:Immunoblotting; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; D003561:Cytarabine; D010768:Phosphoserine; D002110:Caffeine; D005047:Etoposide; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D010744:Phosphoric Monoester Hydrolases; D002945:Cisplatin; D006918:Hydroxyurea