Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation.
BACKGROUND
The DNA damage checkpoint is a protein kinase-based signaling system that detects and signals physical alterations in DNA. Despite having identified many components of this signaling cascade, the exact mechanisms by which checkpoint kinases are activated after DNA damage, as well as the role of the checkpoint mediators, remain poorly understood.


RESULTS
To elucidate the mechanisms that underlie the MEC1 and RAD9-dependent activation of Rad53, the Saccharomyces cerevisiae ortholog of Chk2, we mapped and characterized in vivo phosphorylation sites present on Rad53 after DNA damage by mass spectrometry. We find that Rad53 requires for its activation multisite phosphorylation on a number of typical and atypical Mec1 phosphorylation sites, thus confirming that Rad53 is a direct target of Mec1, the mammalian ATR homolog. Moreover, by using biochemical reconstitution experiments, we demonstrate that efficient and direct phosphorylation of Rad53 by Mec1 is only observed in the presence of purified Rad9, the archetypal checkpoint mediator. We find that the stimulatory activity of Rad9 requires a phospho- and FHA-dependent interaction with Rad53, which allows Rad53 to be recognized as a substrate for Mec1.


CONCLUSIONS
Our results indicate that Rad9 acts as a bona fide signaling adaptor that enables Rad53 phosphorylation by Mec1. Given the high degree of conservation of checkpoint signaling in eukaryotes, we propose that one of the critical functions of checkpoint mediators such as MDC1, 53BP1, or Brca1 is to act as PIKK adaptors during the DNA damage response.
Sweeney
Frédéric D
FD
Centre for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada.
Yang
Feng
F
Chi
An
A
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Durocher
Daniel
D
D001345:Autoradiography; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004789:Enzyme Activation / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae