Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes.
Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, we show here that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes. Moreover, we show that Chk1 activity is not appreciably altered during maturation, being maintained at basal levels, and that C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes. These results, together with other results, suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization. Based on these results, we discuss the possibility that Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and that Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change.
Oe
T
T
Department of Biology, Graduate School of Sciences, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-8581, Japan.
Nakajo
N
N
Katsuragi
Y
Y
Okazaki
K
K
Sagata
N
N
D048948:14-3-3 Proteins; D000818:Animals; D002451:Cell Compartmentation; D000071877:Checkpoint Kinase 1; D003593:Cytoplasm / Q000378:metabolism*; D005260:Female; D016195:G2 Phase / Q000502:physiology; D008954:Models, Biological; D009154:Mutation; D009865:Oocytes / Q000502:physiology*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins; D020687:cdc25 Phosphatases / Q000378:metabolism*
D048948:14-3-3 Proteins; D010446:Peptide Fragments; D029867:Xenopus Proteins; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D020687:cdc25 Phosphatases