Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ

Figure 3 Amino terminal serine cluster (Ser14, Ser17, Ser18, Ser19) of Ankrd54 is required for phorbol 12-myristate 13-acetate stimulated nuclear export. A: Localization analysis of eGFP-tagged Ankrd54 (green) in HEK293 cells stimulated with PMA (200 nmol/L) for the indicated times. Delineation of the nucleus by Hoechst staining (blue), and F-actin with TRITC-phalloidin (red). Nuclear and cytoplasmic localization of Ankrd54 was enumerated (graph at right), ^bP < 0.01, ns: Not significant (P > 0.05); B-E: Localization analysis of eGFP-tagged Ankrd54 by immunofluorescence of cells (left panels) and by Phos-Tag™ SDS-PAGE (pTag) analysis (right panel), in HEK293 cells with and without PMA treatment (200 nmol/L, 60 min). Delineation of the nucleus by Hoechst staining (blue), and F-actin with TRITC-phalloidin (red). Nuclear and cytoplasmic localization of Ankrd54 was enumerated (graph in centre), ^bP < 0.01. Right panel, immunoblot analysis of nuclear (N) and cytoplasmic (C) fractionation of transfected HEK293 cells were immunoblotted using anti-Ankrd54, anti-Histone (H3), anti-14-3-3ζ, and anti-β-actin antibodies. Mobility shifted Ankrd54, due to phosphorylation, is indicated (pAnkrd54). Cell immunofluorescence and immunoblot analysis was undertaken for cells expressing wild-type (B), serine-alanine (S14A, S17A, S18A, S19A) mutated (C), NLS deleted (D) and NES deleted (E) constructs of eGFP-tagged Ankrd54. For panel (C) a direct in-gel comparison of wild-type and SA mutant Ankrd54, before and after PMA stimulation is shown in the far-right panel. PMA: Phorbol 12-myristate 13-acetate.