Regulation of mitotic function of Chk1 through phosphorylation at novel sites by cyclin-dependent kinase 1 (Cdk1).
Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.
Shiromizu
Takashi
T
Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.
Goto
Hidemasa
H
Tomono
Yasuko
Y
Bartek
Jiri
J
Totsukawa
Go
G
Inoko
Akihito
A
Nakanishi
Makoto
M
Matsumura
Fumio
F
Inagaki
Masaki
M
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D016466:CHO Cells; D000071877:Checkpoint Kinase 1; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays
D011994:Recombinant Proteins; D012694:Serine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; D015739:Nocodazole