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Differential regulation of CLC anion channel by SPAK kinase ortholog mediated multisite phosphorylation
Author(s) -
Miyazaki Hiroaki,
Strange Kevin
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.695.3
Subject(s) - dephosphorylation , phosphorylation , wild type , kinase , microbiology and biotechnology , mutant , linker , chemistry , cytoplasm , biochemistry , biology , phosphatase , gene , computer science , operating system
The CLC channel CLH‐3b is activated and inhibited by cell swelling and shrinkage, respectively, and by changes in meiotic cell cycle progression. Channel inhibition requires concomitant phosphorylation of S742 and S747 mediated by the SPAK ortholog GCK‐3. Cell swelling‐induced dephosphorylation of either S742 or S747 alone by PP1 activates CLH‐3b. Glutamate substitution fully mimics phosphorylation. However, the S747E mutant activates and inactivates in response to cell volume changes much more slowly than either wild type or S742E channels. The slower activation/inactivation of the S747E mutant is not due to altered rates of PP1 mediated dephosphorylation or rates of dephosphorylation‐induced conformational changes. GCK‐3 binds to a 4 amino acid motif located on the channel C‐terminus between the CBS1 and CBS2 domains. The 176 amino inter‐CBS “linker” also contains S742 and S747. Co‐expression of wild type CLH‐3b and GCK‐3 with wild type, S742E or S747E linkers titrates GCK‐3 away from full length channels. However, channels co‐expressed with the S747E linker exhibit altered regulation compared to channels expressed with wild type or S742E linkers. Our data suggest that the phosphorylation state of S742 and S747 may modulate GCK‐3/channel interactions. Such modulation may allow distinct modes of channel regulation under different physiological conditions and may also play a role in GCK‐3 regulation.