Fig. 3. Ski enhances phosphorylation of TAZ by Lats2.

(A) Ski enhanced the kinase activity of Lats2. Myc-Lats2 was immunoprecipitated from 293T cells expressing Myc-Lats2 alone or together with Mst2 and Ski as indicated and subjected to in vitro kinase assay using GST-TAZ as a substrate. The abundance of GST-TAZ, Myc-Lats2, and Flag-Mst2 was measured by Western blotting (lower panels). Western blots are representative of three independent experiments. ³²P-TAZ abundance was quantified from at least three independent experiments, and data are presented as means ± SEM (Student’s t test, *P < 0.05). (B) Endogenous Lats2 was immunoprecipitated from MCF10A cells stably expressing control vector, Ski, and shSki and subjected to an in vitro kinase assay with GST-TAZ as an exogenous substrate. The abundance of GST-TAZ and Lats2 was measured by Western blotting. Western blots are representative of three independent experiments. Data are presented as means ± SEM (Student’s t test, *P < 0.05). (C) Pulse-chase assays. ³⁵S-labeled Flag-TAZ was immunoprecipitated with anti-Flag beads and detected by autoradiography. Data in the graph were derived from at least three independent experiments and are presented as means ± SEM (Student’s t test, *P < 0.05). (D and E) Western blot analysis indicated that endogenous TAZ abundance was enhanced in shSki-expressing cells (D) and reduced in Ski over-expressing cells (E). Western blots are representative of three independent experiments. (F) Ski enhanced the Lats2-Sav interaction. HA-Sav was immunoprecipitated from cells transfected with fixed amounts of Myc-Lats2 and HA-Sav and increasing amounts of Flag-Ski, and the associated Myc-Lats2 was detected by Western blotting with anti-Myc antibody. Cells transfected with Flag-Ski and Myc-Lats2 were used as negative controls for anti-Myc immunoprecipitation. Western blots are representative of three independent experiments. (G) Reducing Lats2 by siRNA reversed the inhibition of TAZ by Ski in 3D morphogenesis. Top: Phase-contrast images of 3D acini. Bottom: Forty acini from three independent experiments were quantified for each cell line and presented in the graph as means ± SEM (ANOVA, Newman-Keuls multiple comparison test). Significant differences between TAZ+Ski cells expressing control vector and Lats2 siRNA (siLats2) were detected (**P < 0.05). Scale bar, 40 μm. (H) Reducing Lats2 reversed the Ski inhibition of TAZ-dependent actin stress fiber formation. Images are representative of three independent experiments. Scale bar, 20 μm.