Active Gwl in the cytoplasm is necessary and sufficient for the induction of Endos phosphorylation and interaction with PP2A-Tws. (A) Mutations in Gwl used to alter its kinase activity and localization. Top: Location of the mutations in the primary structure. Middle: IF showing the localization of the Gwl-Myc variants. Bottom: Subcellular fractionation and WB showing the relative amounts of cytoplasmic and nuclear proteins. MEK and histone H3 are controls as cytoplasmic and nuclear proteins, respectively. Scale bar: 5 μm. (B) Expression of the constitutively active and cytoplasmic Gwl^{K97M, NLSm}-Myc increases levels of pS68-Endos. Cells were transfected with the indicated constructs, treated with 100 nM Okadaic acid for 1 h, and analyzed by WB. (C) Expression of the constitutively active cytoplasmic Gwl^{K97M, NLSm}-Myc enhances the interaction between Endos and Tws. Cells were transfected with the indicated constructs and submitted to Tws immunoprecipitation (IP). Products were analyzed by WB. *, IgG. C, cytoplasmic; Dm, Drosophila melanogaster; N, nuclear; NES, nuclear export signal; ter., terminal; WCE, whole-cell extract.