Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.
Guillemain
Ghislaine
G
CEA, Direction des Sciences du Vivant, Institut de Biologie et de la Technologies de Saclay, 91191 Gif-sur-Yvette Cedex, France.
Ma
Emilie
E
Mauger
Sarah
S
Miron
Simona
S
Thai
Robert
R
Guérois
Raphaël
R
Ochsenbein
Françoise
F
Marsolier-Kergoat
Marie-Claude
MC
D000220:Adaptation, Biological; D000483:Alleles; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D009682:Magnetic Resonance Spectroscopy; D010748:Phosphopeptides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D010748:Phosphopeptides; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D064447:Checkpoint Kinase 2; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C491716:PTC2 protein, S cerevisiae; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases