Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1.
Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12). Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant. Cds1(T11A) was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.
Tanaka
K
K
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Boddy
M N
MN
Chen
X B
XB
McGowan
C H
CH
Russell
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004265:DNA Helicases / Q000378:metabolism*; D004789:Enzyme Activation; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D013912:Threonine
D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D013912:Threonine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases