Hedgehog signal transduction: recent findings

The Hedgehog (Hh) family of signaling molecules are key agents in patterning numerous types of tissues. Mutations in Hh and its downstream signaling molecules are also associated with numerous oncogenic and disease states. Consequently, understanding the mechanisms by which Hh signals are transduced is important for understanding both development and disease. Recent studies have clarified several aspects of Hh signal transduction. Several new Sonic Hedgehog binding partners have been identified. Cholesterol and palmitic acid modifications of Hh and Sonic hedgehog have been examined in greater detail. Characterization of the trafficking patterns of the Patched and Smoothened proteins has demonstrated that these two proteins function very differently from the previously established models. The Fused kinase has been demonstrated to phosphorylate the kinesin-like protein Costal2 and the sites identified, while Cubitus interruptus has been shown to be phosphorylated in a hierarchical manner by three different kinases. Finally, the interactions, both genetic and physical, between Fused, Costal2, Cubitus interruptus, and Suppressor of Fused have been further elucidated.     

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.     

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.     

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.     

The sex determination master switch, Sex-lethal, responds to Hedgehog signaling in the Drosophila germline

Sex-lethal is the Drosophila melanogaster sex determination master switch. It is also required in female germ cells to control mitosis and meiotic recombination. As early germ cells mature, distinct changes in both Sex-lethal protein levels and localization occur. By manipulating the levels of Hedgehog and making germline clones of components in the hedgehog signaling pathway, we demonstrate that Hedgehog affects the nuclear translocation of Sex-lethal and the levels of the protein in early germ cells. This effect is mediated primarily through degradation. Consistent with the Hedgehog pathway regulating Sex-lethal, we find Sex-lethal in a complex with Fused and Costal-2, both downstream components of the pathway. This is the first demonstration that downstream components of the Hedgehog signaling pathway regulate a target other than Cubitus interruptus.     

A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci.