Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells

被引:29
作者
Charych, Deborah H. [1 ]
Coyne, Mazie [1 ]
Yabannavar, Asha [1 ]
Narberes, Jamie [1 ]
Chow, Sylvia [1 ]
Wallroth, Marco [1 ]
Shafer, Cynthia [1 ]
Walter, Annette O. [1 ]
机构
[1] Novartis Inst Biomed Res, Emeryville, CA 94608 USA
关键词
kinase; phosphorylation site; mass spectrometry; cancer; target modulation;
D O I
10.1002/jcb.21698
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNAdamage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.
引用
收藏
页码:1075 / 1086
页数:12
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