title: Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells.
abstract: Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. arai@idaten.c.u-tokyo.ac.jp
authors.forename: Hideaki
authors.initials: H
authors.lastname: Arai
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Atomi
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D051339:Actin Depolymerizing Factors; D021961:Cell Membrane Structures / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D054461:Lim Kinases; D008840:Microfilament Proteins / Q000187:drug effects / Q000378:metabolism*; D010759:Phosphorus Isotopes; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D051339:Actin Depolymerizing Factors; D004268:DNA-Binding Proteins; D007328:Insulin; D008840:Microfilament Proteins; D010759:Phosphorus Isotopes; D011494:Protein Kinases; C515892:LIMK1 protein, human; D054461:Lim Kinases; D017346:Protein Serine-Threonine Kinases