Polo interacts with and phosphorylates Gwl. (A) Polo is detected by Western blot in a Gwl-PrA purification product from embryos. (B) Polo-Myc is copurified with Gwl-PrA from transgenic embryos. (C) The association of Polo with Gwl does not depend on its canonical phospho-binding activity. D-Mel cells expressing the indicated proteins were used in PrA affinity purifications followed by Western blots. Mut = Polo W395F, H538A, K540A. (D) The central region of Gwl associates with Polo. Experiment done as in C. (E) Polo phosphorylates Gwl in vitro. KD: GST-Gwl-K87R, kinase-dead. BI2536 is a Polo inhibitor that was used as a control. (F) Moderate overexpression of Polo-Myc in embryos induces hyperphosphorylation of Gwl. (G) Gwl hyperphosphorylation depends on Polo in embryo extracts. After lysis, extracts were incubated as indicated. Okadaic acid induces an upshift in the mobility of Gwl that is abrogated by BI2536 (200 nM). (H) Model: Polo interacts with Gwl in its central region and phosphorylates multiple sites.