Cdc5 blocks in vivo Rad53 activity, but not in situ activity (ISA).
DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation, but does not have the auto-phosphorylations required for full activity towards exogenous substrates.
Lopez-Mosqueda
Jaime
J
Dept. of Biochemistry and Biophysics, University of California, San Francisco, USA.
Vidanes
Genevieve M
GM
Toczyski
David P
DP
D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D017403:In Situ Hybridization; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; C067022:RAD53 protein, S cerevisiae