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.