Identification of a tetrameric hedgehog signaling complex

Hedgehog (Hh) signal transduction requires a large cytoplasmic multi-protein complex that binds microtubules in an Hh-dependent manner. Here, we show that three members of this complex, Costal2 (Cos2), Fused (Fu), and Cubitus interruptus (Ci), bind each other directly to form a trimeric complex. We demonstrate that this trimeric signaling complex exists in Drosophila lacking Suppressor of Fused (Su(fu)), an extragenic suppressor of fu, indicating that Su(fu) is not required for the formation, or apparently function, of the Hh signaling complex. However, we subsequently show that Su(fu), although not a requisite component of this complex, does form a tetrameric complex with Fu, Cos2, and Ci. This additional Su(fu)-containing Hh signaling complex does not appear to be enriched on microtubules. Additionally, we demonstrate that in response to Hh Ci accumulates in the nucleus without its various cytoplasmic binding partners, including Su(fu). We discuss a model in which Su(fu) and Cos2 each bind to Fu and Ci to exert some redundant effect on Ci such as cytoplasmic retention. This model is consistent with genetic data demonstrating that Su(fu) is not required for Hh signal transduction proper and with the elaborate genetic interactions observed among Su(fu), fu, cos2, and ci.     

Hedgehog signaling downregulates Suppressor of Fused through the HIB/SPOP-Crn axis in Drosophila

Hedgehog (Hh) signaling plays vital roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and several types of cancer. Suppressor of Fused (Su(fu)) is a conserved inhibitory component of the Hh signaling pathway, but how it is regulated remains poorly understood. Here we demonstrate that in Drosophila Hh signaling promotes downregulation of Su(fu) through its target protein HIB (Hh-induced BTB protein). Interestingly, although HIB-mediated downregulation of Su(fu) depends on the E3 ubiquitin ligase Cul3, HIB does not directly regulate Su(fu) protein stability. Through an RNAi-based candidate gene screen, we identify the spliceosome factor Crooked neck (Crn) as a regulator of Su(fu) level. Epistasis analysis indicates that HIB downregulates Su(fu) through Crn. Furthermore, we provide evidence that HIB retains Crn in the nucleus, leading to reduced Su(fu) protein level. Finally, we show that SPOP, the mammalian homologue of HIB, can substitute HIB to downregulate Su(fu) level in Drosophila. Our study suggests that Hh regulates both Ci and Su(fu) levels through its target HIB, thus uncovering a novel feedback mechanism that regulates Hh signal transduction. The dual function of HIB may provide a buffering mechanism to fine-tune Hh pathway activity.     

Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms

The Hedgehog (Hh) family of secreted proteins governs many developmental processes in both vertebrates and invertebrates. In Drosophila, Hh acts by blocking the formation of a truncated repressor form of Cubitus interruptus (Ci) and by stimulating the nuclear translocation and activity of full-length Ci (Ci155). In the absence of Hh, Ci155 is sequestered in the cytoplasm by forming protein complexes with Costal2 (Cos2), Fused (Fu) and Suppressor of Fused [Su(fu)]. How complex formation regulates Ci155 subcellular localization is not clear. We find that Cos2 interacts with two distinct domains of Ci155, an amino (N)-terminal domain (CDN) and a carboxyl (C)-terminal domain (CORD), and Cos2 competes with Su(fu) for binding to the N-terminal region of Ci155. We provide evidence that both N- and C-terminal Cos2 binding domains are involved in the cytoplasmic retention of Ci155 in imaginal discs. Treating imaginal discs with microtubule-destabilizing reagent nocodazole promotes nuclear translocation of Ci155, suggesting that the microtubule network plays an important role in the cytoplasmic retention of Ci155. In addition, we find that adding a nuclear localization signal (NLS) to exposed regions of Ci155 greatly facilitates its nuclear translocation, suggesting that the cytoplasmic retention of Ci155 may also depend on NLS masking.     

Transduction of the Hedgehog signal through the dimerization of Fused and the nuclear translocation of Cubitus interruptus

The Hedgehog (Hh) family of secreted proteins is essential for development in both vertebrates and invertebrates. As one of main morphogens during metazoan development, the graded Hh signal is transduced across the plasma membrane by Smoothened (Smo) through the differential phosphorylation of its cytoplasmic tail, leading to pathway activation and the differential expression of target genes. However, how Smo transduces the graded Hh signal via the Costal2 (Cos2)/Fused (Fu) complex remains poorly understood. Here we present a model of the cell response to a Hh gradient by translating Smo phosphorylation information to Fu dimerization and Cubitus interruptus (Ci) nuclear localization information. Our findings suggest that the phosphorylated C-terminus of Smo recruits the Cos2/Fu complex to the membrane through the interaction between Smo and Cos2, which further induces Fu dimerization. Dimerized Fu is phosphorylated and transduces the Hh signal by phosphorylating Cos2 and Suppressor of Fu (Su(fu)). We further show that this process promotes the dissociation of the full-length Ci (Ci155) and Cos2 or Su(fu), and results in the translocation of Ci155 into the nucleus, activating the expression of target genes.