The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1delta/epsilon.
Dnmt1 (DNA methyltansferase 1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we investigated protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. A protein fraction containing protein kinase activity for phosphorylation of Dnmt1(1-290) was prepared using Dnmt1(1-290)-affinity, DNA-cellulose and gel-filtration columns. When the proteins in this fraction were analysed by LC-MS/MS (liquid chromatography tandem MS), CK1delta/epsilon (casein kinase 1delta/epsilon) was the only protein kinase identified. Recombinant CK1delta/epsilon was found to bind to the N-terminal domain of Dnmt1 and significantly phosphorylated this domain, especially in the presence of DNA. Phosphorylation analyses using various truncation and point mutants of Dnmt1 revealed that the major priming site phosphorylated by CK1delta/epsilon was Ser146, and that subsequent phosphorylation at other sites may occur after phosphorylation of the priming site. When the DNA-binding activity of phosphorylated Dnmt1 was compared with that of the non-phosphorylated form, phosphorylation of Dnmt1 was found to decrease the affinity for DNA. These results suggest that CK1delta/epsilon binds to and phosphorylates the N-terminal domain of Dnmt1 and regulates Dnmt1 function by reducing the DNA-binding activity.
Sugiyama
Yasunori
Y
Department of Life Sciences, Kagawa University, Japan.
Hatano
Naoya
N
Sueyoshi
Noriyuki
N
Suetake
Isao
I
Tajima
Shoji
S
Kinoshita
Eiji
E
Kinoshita-Kikuta
Emiko
E
Koike
Tohru
T
Kameshita
Isamu
I
D000818:Animals; D015153:Blotting, Western; D048149:Casein Kinase 1 epsilon / Q000378:metabolism*; D048148:Casein Kinase Idelta / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004247:DNA / Q000378:metabolism*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D053719:Tandem Mass Spectrometry
D004247:DNA; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse; D048149:Casein Kinase 1 epsilon; D048148:Casein Kinase Idelta