Casein kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1.
The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded by the Rad17-RFC complex onto chromatin after DNA damage and plays a key role in the ATR-dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9-1-1 and phosphorylates serines 341 and 387 (Ser-341 and Ser-387) in the C-terminal tail of Rad9. Interestingly, phosphorylated Ser-387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9-1-1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over-expressed in HeLa cells, a mutant Rad9 harboring phospho-deficient substitutions at both Ser-341 and Ser-387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR-dependent checkpoint pathway through its ability to phosphorylate Ser-341 and Ser-387 of the Rad9 subunit of the 9-1-1 complex.
Takeishi
Yukimasa
Y
Department of Biology, Kyushu University, Higashi-ku, Fukuoka, Japan.
Ohashi
Eiji
E
Ogawa
Kaori
K
Masai
Hisao
H
Obuse
Chikashi
C
Tsurimoto
Toshiki
T
D002352:Carrier Proteins / Q000378:metabolism*; D047390:Casein Kinase II / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C549605:HUS1 protein, human; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; C069930:rad9 protein; D012694:Serine; D047390:Casein Kinase II; D005092:Exonucleases; C113574:Rad1 protein, human