Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae.
Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage check-point kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.
Chen
Sheng-hong
SH
Division of Biological Sciences, Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla 92093-0653, USA.
Smolka
Marcus B
MB
Zhou
Huilin
H
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D017124:Conserved Sequence; D004249:DNA Damage / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D000071164:Trefoil Factor-2
D018797:Cell Cycle Proteins; D009077:Mucins; D009124:Muscle Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C481546:TFF2 protein, mouse; D000071164:Trefoil Factor-2; D013912:Threonine; D011494:Protein Kinases; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; C578633:Chek2 protein, mouse; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae