Dissection of Mechanisms Regulating the Drosophila Hedgehog Pathway

Hedgehog (Hh) signaling is essential for both embryonic development and adult tissue homeostasis. Malfunction of Hh signaling pathway causes many human disorders including birth defects and cancers. In Drosophila, the G-protein-coupled-receptor-like protein Smoothened (Smo) transduces the Hh signal across the plasma membrane, and an intracellular Hh signaling complex (HSC) containing the kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu) and a PEST-domain containing protein suppressor of Fused (Sufu) relays the Hh signal downstream from Smo to the Zn finger transcription factor Cubitus interruptus (Ci). Our previous studies have demonstrated that Hh transduces signal by regulating the subcellular localization and conformational state of Smo, but how Smo relays the signal to cytoplasmic signaling components remains poorly understood. In this study, we show that Hh-induced Smo conformational change promotes the recruitment of Cos2/Fu complex and Fu dimerization. We find that induced dimerization through the Fu kinase domain activates Fu by inducing multi-site phosphorylation of its activation loop (AL), and phospho-mimetic mutations of AL suffice to activate the Hh pathway. Moreover, we find that activated Fu regulates Ci by both promoting its transcriptional activator activity and inhibiting its proteolysis into a repressor form. We provide evidence to suggest that activated Fu exerts the regulation by interfering with the formation of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. In the rest part of the study, we further explore additional mechanisms regulating Ci activity. We have identified and characterized three types of functional regulatory elements in Ci, including a transcriptional repression domain in the N-terminal region of Ci, multiple Ser/Thr motifs in the amino-(N-) and carboxy-(C-) terminal regions of Ci serving as HIB/SPOP E3 ligase-specific degrons, and finally a novel PY-NLS around the N-terminal highly conserved domain of Ci.