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Phosphorylation and functional regulation of CIC-2 chloride channels expressed in Xenopus oocytes by M cyclin-dependent protein kinase

By Tetsushi Furukawa, Takehiko Ogura, Ya-Juan Zheng, Hiroyuki Tsuchiya, Haruaki Nakaya, Yoshifumi Katayama and Nobuya Inagaki

Abstract

Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl− channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume-sensitive Cl− channel, ClC-2 is regulated by the M-phase-specific cyclin-dependent kinase, p34cdc2/cyclin B. ClC-2 channels were phosphorylated by p34cdc2/cyclin B in both in vitro and cell-free phosphorylation assays. ClC-2 phosphorylation was inhibited by olomoucine and abolished by a 632Ser-to-Ala (S632A) mutation in the C-terminus, indicating that 632Ser is a target of phosphorylation by p34cdc2/cyclin B. Injection of activated p34cdc2/cyclin B attenuated the ClC-2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC-2 currents attenuated by p34cdc2/cyclin B were increased by application of the cyclin-dependent kinase inhibitor, olomoucine (100 μm), an effect that was inhibited by calyculin A (5 nm) but not by okadaic acid (5 nm). A yeast two-hybrid system revealed a direct interaction between the ClC-2 C-terminus and protein phosphatase 1. These data suggest that the ClC-2 channel is also counter-regulated by protein phosphatase 1. In addition, p34cdc2/cyclin B decreased the magnitude of ClC-2 channel activation caused by cell swelling. As the activities of both p34cdc2/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC-2 channel could be regulated physiologically by these factors

Topics: Research Papers
Publisher: Blackwell Science Inc
OAI identifier: oai:pubmedcentral.nih.gov:2290285
Provided by: PubMed Central
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