Transient transfection of NIH3T3 cells with various constructs of myosin phosphatase target subunit (MYPT1) and GFP showed distinct cellular localizations. Constructs containing the N-terminal nuclear localization signals (NLS), i.e. full-length MYPT1 and N-terminal MYPT1 fragments, were concentrated in the nucleus. Full-length chicken and human MYPT1-GFP showed discrete nuclear foci. Deletion of the N-terminal NLS or use of central or C-terminal MYPT1 fragments did not show unique nuclear distributions (C-terminal NLS are present). Transient transfection of NIH3T3 cells (in the presence of serum) with full-length MYPT1-GFP caused a marked decrease in number of attached cells, an apparent block in the cell cycle prior to M phase and signs of increased apoptosis. Under conditions of serum starvation the unique nuclear localization of MYPT1-GFP was not found and there was no marked decrease in the number of attached cells (after 48 h). Stable transfection of HEK 293 cells with GFP-MYPT1 was obtained. MYPT1 and its N-terminal mutants bound to retinoblastoma protein (Rb), raising the possibility that Rb is implicated in the effects caused by overexpression of MYPT1.
ASJC Scopus subject areas
- Cell Biology