Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast.
Genome integrity is monitored by a checkpoint that delays mitosis in response to DNA damage. This checkpoint is enforced by Chk1, a protein kinase that inhibits the mitotic inducer Cdc25. In fission yeast, Chk1 is regulated by a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR. These kinases phosphorylate serine or threonine followed by glutamine (SQ/TQ). Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367. Serine-345 of human Chk1 is phosphorylated in response to DNA damage. Here we report that Rad3 and ATM phosphorylate serine-345 of fission yeast Chk1. Mutation of serine-345 (chk1-S345A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo. The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective. In contrast, mutations of serine-367 and other SQ/TQ sites do not substantially impair the checkpoint or cause damage sensitivity. These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen evidence that transduction of the DNA damage checkpoint signal requires direct phosphorylation of Chk1 by Rad3.
Lopez-Girona
A
A
Department of Molecular and Cell Biology, MB3, The Scripps Research Institute, La Jolla, CA 92037, USA.
Tanaka
K
K
Chen
X B
XB
Baber
B A
BA
McGowan
C H
CH
Russell
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D017124:Conserved Sequence; D004265:DNA Helicases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011898:Ranidae; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012694:Serine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; C000605873:Chk1 protein, S pombe; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases