Fig. 5.

Shh signaling-induced Inversin phosphorylation by PKA increases Inversin–Smo interaction. (A) Inversin interacts with Smo, and its T794 phosphorylation increases this interaction. HEK 293T cells were cotransfected with the indicated plasmids, arrested at the G0 phase, treated with or without FSK, and processed to immunoprecipitation assays. Note that losing the ability of T794 phosphorylation of Inversin by PKA impaired its binding with Smo. (B) Smo interacts with Inversin at the ciliary proximal region in Ptc1^−/− cells. G0 phase Ptc1^−/− cells were subjected to the PLA assay (see details in SI Appendix, Materials and Methods), singly labeled with Smo or Inversin, or colabeled with Smo and Inversin. (Scale bar, 5 μm.) (C and D) The phosphorylation-mimicking mutant of Inversin recruits Smo to the ciliary proximal region. WT cells stably expressing the WT or T794A/D mutant GFP-Inversin were arrested at the G0 phase and immunostained for the indicated proteins. The percentage of PC-patterned Smo in each cell line is shown in D. In each of three independent experiments, 50 cells per sample were randomly selected and counted. ***P < 0.001. (E) The T794D mutant Inversin results in a partial activation of Shh pathway. The parental cells (WT and Ptc1^−/−) and the cells stably expressing the WT, T794A/D GFP-Inversin (labeled in green) were arrested at the G0 phase and examined for the indicated proteins. (F) Smo–Inversin interaction is increased by Shh pathway activation. HEK 293T cells were cotransfected with the indicated plasmids, arrested at the G0 phase, and processed to immunoprecipitation assays. The normalized results of total and immunoprecipitated upper band of Smo-Myc by the indicated proteins are shown Below each panel, and the amount of Smo-Myc under normal condition (UNT) was set as 1.0.