M phase-specific expression and phosphorylation-dependent ubiquitination of the ClC-2 channel.
Cl(-) channel activities vary during the cell cycle and are thought to play various roles including regulation of cell volume. We have shown previously that ClC-2 channels are directly phosphorylated and functionally regulated by the M phase-specific cyclin-dependent kinase p34(cdc2)/cyclin B. We investigate here to determine whether the expression levels of ClC-2 channel protein vary during the cell cycle. Immunoblot and immunocytochemical analyses of cells cycle-synchronized by serum depletion/replenishment reveal that ClC-2 channel protein is expressed predominantly at M phase in cells with two nuclei and a clear constriction ring, whereas RNA blot analysis shows that ClC-2 mRNA expression does not change during the cell cycle. Ubiquitin assays reveal that the ClC-2 channels are ubiquitinated at M phase, whereas the magnitude of ubiquitination is suppressed by incubation with olomoucine, an inhibitor of p34(cdc2)/cyclin B, and it is almost completely abolished in ClC-2 channels having an S632A mutation, which cannot be phosphorylated by p34(cdc2)/cyclin B, indicating that ubiquitination of ClC-2 channels requires phosphorylation by M phase-specific p34(cdc2)/cyclin B. Regulation at the post-transcriptional level, including phosphorylation-dependent ubiquitination, may contribute to M phase-specific expression of ClC-2 channels. Cell cycle-dependent regulation of expression at the protein level in addition to the regulation of function suggests that the ClC-2 channel plays a physiological role in the cell cycle.
Zheng
Ya-Juan
YJ
Department of Physiology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.
Furukawa
Tetsushi
T
Ogura
Takehiko
T
Tajimi
Kimitaka
K
Inagaki
Nobuya
N
D000818:Animals; D000090007:CLC-2 Chloride Channels; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D006321:Heart / Q000502:physiology*; D006801:Humans; D008938:Mitosis / Q000502:physiology; D009206:Myocardium / Q000166:cytology*; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D000090007:CLC-2 Chloride Channels; D015220:Calcium Channels; D018118:Chloride Channels; D011994:Recombinant Proteins; D025801:Ubiquitin