Saccharomyces cerevisiae Rad53, the ortholog of mammalian Chk2, is an essential protein kinase in DNA damage and DNA replication checkpoint pathways. Consecutive phosphatidyl inositol kinase-like kinase (PIKK)-dependent and PIKK-independent steps in activation of Rad53 are key steps for controlling and transmitting diverse downstream responses to DNA damage. However, these activities have not been demonstrated in vitro in defined systems. Here, we have shown that enzymatically dephosphorylated purified Rad53 autoactivates in vitro through a phosphorylation-dependent mechanism. Kinetic analysis demonstrated that autophosphorylation results in a more than 9-fold increase in protein kinase activity. Autophosphorylation was Rad53 concentration-dependent, indicating that the reaction follows an intermolecular mechanism. DNA damage induced oligomerization of a subset of Rad53 molecules in vivo. At low concentrations of Rad53, preincubation of Rad53 with immune complexes containing the Mec1/Ddc2 complex can activate Rad53 kinase activity. Our findings showed that Mec1/Ddc2 complexes can directly activate Rad53 through a phosphorylation-dependent mechanism, and more generally, supported the hypothesis that PIKKs regulate Chk2 orthologs through phosphorylation. Moreover, this work has substantiated a model for PIKK-independent amplification of Rad53 activation (and by extension, activation of other Chk2 orthologs) mediated by inter-Rad53 phosphorylation.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|