| Research Abstract |
Scapinin has been identified as a nuclear structure protein that binds and inhibits protein phosphatase-1 (PP1) in a leukemia cell line HL-60. We have also noticed several discrepancies against ideas that scapinin is a constitutive component of the nonchromatin structure of leukocytes. In adult tissues, scapinin is highly expressed in brain but not peripheral polynuclear leukocytes. Furthermore, scapinin was localized in cytoplasmic protrusions, so called microvilli, in addition to the nucleus. In this study, we executed several approaches to identify scapinin-binding proteins ; yeast two-hybrid, mass spectrometric analyses of polypeptides co-immunoprecipitated with scapinin from HL-60 cell lysate, and pull-down assay with GST-scapinin fusion protein conjugated agarose beads. Both two-hybrid and mass spectrometric analyses demonstrated that actin is a binding protein of scapinin, and the GST-scapinin fusion proteins showed that the REPL repeat, which is a 〜twenty-amino acids motif with conserved arginine, glutamic acid, proline, and leucine residues, are responsible to the actin binding ability. We established a cell line that express scapinin under control of tetracycline. Tet-induced expression of scapinin enhanced cell adhesion, and, consistently, scapinin localized at tips of cell adhesion, lamellapodia, in which actin nearly accumulated. These observations demonstrated that scapinin modulates actin structure and enhances cell adhesion. Two-hybrid assay also showed candidates for scapinin-interacting proteins that were implicated in regulation of transcription, cell adhesion, or membrane receptor. Further studies are needed for conclusions.
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