title: Activity of cofilin can be regulated by a mechanism other than phosphorylation/dephosphorylation in muscle cells in culture.
abstract: Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP(2). To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP(2) can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP(2) in the cytoplasm of muscle cells.
authors.affiliation: Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hosoda
authors.forename: Naruki
authors.initials: N
authors.lastname: Sato
authors.forename: Rie
authors.initials: R
authors.lastname: Nagaoka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Abe
authors.forename: Takashi
authors.initials: T
authors.lastname: Obinata
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D051338:Cofilin 1 / Q000378:metabolism* / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism