The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation. Qing Shi¹, Shuang Li¹, Jianhang Jia², Jin Jiang¹. 1) Department of Developmental Biology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA; 2) Markey Cancer Center and Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.

   Hedgehog (Hh) transduces signal by regulating the subcellular localization and conformational state of the GPCR-like protein Smoothened (Smo) but how Smo relays the signal to cytoplasmic signaling components remains poorly understood. Here, by colocalization study and fluorescence resonance energy transfer (FRET) analysis, we find that Hh-induced Smo conformational change recruits Costal2 (Cos2)/Fused (Fu) and promotes Fu kinase domain dimerization. By performing mutagenesis analysis and using phospho-specific antibodies, we show that induced dimerization through the Fu kinase domain activates Fu by inducing multi-site phosphorylation of its activation loop (AL) and that phospho-mimetic mutations of AL activate the Hh pathway. Interestingly, we observe that graded Hh signals progressively increase Fu kinase domain dimerization and AL phosphorylation, suggesting that Hh activates Fu in a dose-dependent manner. Moreover, we find that activated Fu regulates Cubitus interruptus (Ci) by both promoting its transcriptional activator activity and inhibiting its proteolysis into a repressor form. We provide evidence that activated Fu exerts these regulations by interfering with the formation of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. Taken together, our results suggest that Hh-induced Smo conformational change facilitates the assembly of active Smo-Cos2-Fu signaling complexes that promote Fu kinase domain dimerization, phosphorylation and activation, and that Fu regulates both the activator and repressor forms of Ci.