title: Novel PKCα-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release.
abstract: Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCβI, we find that PKCβI positively regulates degranulation 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.
authors.affiliation: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Sakuma
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Shirai
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yoshino
authors.forename: Maho
authors.initials: M
authors.lastname: Kuramasu
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Yanagita
authors.forename: Kensaku
authors.initials: K
authors.lastname: Mizuno
authors.forename: Izumi
authors.initials: I
authors.lastname: Hide
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakata
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015550:Cell Degranulation; D045744:Cell Line, Tumor; D051338:Cofilin 1 / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006636:Histamine Release; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D058105:Polymerization; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D000199:Actins; C495812:Cfl1 protein, rat; D051338:Cofilin 1; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D049452:Green Fluorescent Proteins; D012694:Serine; C099154:ruboxistaurin; D051571:Protein Kinase C-alpha