Figure 7. Daple suppresses proliferation and tumorigenesis via its GBA motif.
(A) Daple's GBA motif is required for inhibition of cell transformation induced by oncogenic KRas. NIH3T3 cells stably expressing HA-KRas G12V alone or coexpressing HA-KRas G12V with myc-Daple-WT or various mutants were analyzed for their ability to form colonies in soft agar prior to staining with MTT. The top panel displays representative images of colony-containing plates. Bar graphs in the lower panel shows % inhibition of colony formation (y axis) by each Daple construct compared to NIH3T3 cells transformed with KRas G12V alone. Lysates of NIH3T3 cells were analyzed for Daple and Ras constructs by IB (see Figure 7—figure supplement 1B). (B) Daple is required for inhibition of anchorage-dependent tumor growth by Wnt5a. Control (shLuc) and Daple-depleted (sh Daple 1) HeLa cells were analyzed for their ability to form colonies on plastic plates in the presence (+) or absence (−) of Wnt5a during a 2-week period prior to fixation and staining with crystal violet. Left panel shows the photograph of the crystal violet-stained wells of a 6-well plate. The number of colonies was counted by ImageJ (Colony counter). Right panel shows bar graphs that display the % inhibition of colony formation (y axis) seen in each condition normalized to control (shLuc) HeLa cells. (C) Daple's GBA motif is required for inhibition of anchorage-dependent tumor growth by Wnt5a. Daple-depleted (sh Daple 1) HeLa cells stably expressing either Daple WT or FA were analyzed for their ability to form colonies on plastic plates in the presence (+) or absence (−) of Wnt5a prior to fixation and staining with crystal violet, photographed and analyzed as in B. Left panel shows the photograph of the crystal violet-stained wells of a 6-well plate. Right panel shows bar graphs that display the % inhibition of colony formation (y axis) seen in each condition normalized to control (shLuc) HeLa cells. (D–F) Daple's GBA motif is required for inhibition of anchorage-independent tumor growth. DLD1 cells expressing either control vector or various myc-Daple constructs were analyzed for their ability to form colonies in soft agar for 2–3 weeks. In panel D, representative fields photographed at 20× magnification are shown. The number of colonies was counted by light microscopy throughout the depth of the matrix in 15 randomly chosen fields. In panel E, bar graphs display the number of colonies (y axis) seen in each cell line in D. In panel F, lysates of DLD1 cells used in D were analyzed for Daple constructs by IB. (G, H) Daple's GBA motif is required for inhibition of anchorage-dependent tumor growth. DLD1 cells used in D were analyzed for their ability to form adherent colonies on plastic plates during 2–3 weeks prior to fixation and staining with crystal violet. In panel G, photograph of the crystal violet-stained 6-well plate is displayed. The number of colonies was counted by ImageJ (Colony counter). In panel H, bar graphs display the % inhibition of colony formation (y axis) seen in each cell line in G normalized to control DLD1 cells. (I) Schematic summary. Modulation of G protein activity by Daple's GBA motif is a key determinant of cellular phenotype(s) triggered by Wnt5a. In cells expressing Daple-WT, a functionally intact GBA motif (+) can activate Gαi, enhance PM-based motogenic signals (PI3K-Akt and Rac1 activation), trigger EMT and cell migration/invasion. In cells expression Daple-FA, without the functional GBA motif (−) G protein remains inactive, non-canonical Wnt signaling is suppressed, which increases stability of β-catenin and upregulation of Wnt target genes, resulting in increased transformation, proliferation, and tumor cell growth.
Figure 7—figure supplement 1. Daple suppresses cell proliferation via its GBA motif.
