Novel PKCα-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release

Megumi Sakuma, Yasuhito Shirai, Ken Ichi Yoshino, Maho Kuramasu, Tomofumi Nakamura, Toshihiko Yanagita, Kensaku Mizuno, Izumi Hide, Yoshihiro Nakata, Naoaki Saito

    Research output: Contribution to journalArticlepeer-review

    14 Citations (Scopus)

    Abstract

    Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCαI, we find that PKCβI positively regulates degranula tion in rat basophilic leukemia-2H3 cells, whereas PKCα negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKCα phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form (S23,24E), increases degranulation. Furthermore, the S23,24A mutant binds to F-actin and retains its depolymerizing and/or cleavage activity; conversely, the S23,24E mutant is unable to sever actin filaments, resulting in F-actin polymerization. In addition, the S23,24E mutant preferentially binds to the 14-3-3ζ protein. Fluorescence-activated cell sorting analysis with fluorescein isothiocyanate- phalloidin and simultaneous observation of degranulation, PKC translocation, and actin polymerization reveals that during degranulation, actin polymerization is dependent on PKCα activity. These results indicate that a novel PKCα-mediated phosphorylation event regulates cofilin by inhibiting its ability to depolymerize F-actin and bind to 14-3-3ζ, thereby promoting F-actin polymerization, which is necessary for cessation of degranulation.

    Original languageEnglish
    Pages (from-to)3707-3721
    Number of pages15
    JournalMolecular biology of the cell
    Volume23
    Issue number18
    DOIs
    Publication statusPublished - 2012 Sep 15

    ASJC Scopus subject areas

    • Molecular Biology
    • Cell Biology

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