The protein Hedgehog (Hh) is a highly conserved, secreted ligand (and morphogen) capable of patterning many different tissues during development. Recently, Sonic Hedgehog (SHH) a human homolog of Drosophila Hh was found to be a causative agent in certain cancers. While several drugs are being developed to combat the binding of SHH to its receptor Patched or the Patched-target Smoothened, very little is known about how SHH is secreted from the producing cell, another site for therapeutic targeting. We report here the characterization of a novel proteolytic event and genetic pathway that controls Hh intracellular sorting and axon transport using the Drosophila eye imaginal disc as our model system. In fly larval photoreceptor neurons the developmental signal Hh is guided to the apical (retina) and basal (growth cone, GC) ends where secretion of the morphogen is an inductive factor in photoreceptor differentiation and establishment of eye/brain neural connections. The Hh secreted from the basal side induces lamina development while Hh secreted at the retina induces ommatidial development. Hedgehog processing consists of autocleavage from its 46 kDa form (HhU) to become a lipid-modified N-terminal signaling molecule (HhN; 19kDa) and a C-terminal molecule (HhC24; 24 kDa). Following autocleavage, a fraction of the C-terminal auto-cleavage product then undergoes a second cleavage event leading to 16 kDa (HhC16) and 9 kDa products. Nothing is known about the significance of the C-terminal “2nd cleavage” other than its occurrence in both fly and human tissue. In an effort to identify regulators of Hh sorting, we discovered that the HhC “2nd cleavage” is a determining factor in the sorting of the HhN signaling domain. That is, if a cell induces more cleavage (more HhC16) we observe more HhN in the apical domain. Likewise, if a cell inhibits 2nd cleavage (less HhC16) we see more basal HhN. Creation of a “2nd cleavage mutant” shows that this process has developmental significance. Further, biochemical characterization of the 2nd cleavage suggests it occurs in the ER after autocleavage and that HhC24 can exit the cell in a Golgi independent manner (via lipid droplets) while HhC16 remains intracellular. The ER exit of HhC24 appears to be controlled by a conserved PP2A (Mts) /PKB (Akt) kinase pathway which potentially regulates the size and number of lipid droplets produced. These findings are an important first step in understanding the intracellular sorting and transport of Hh and highlight new targets for the treatment of SHH-related cancers. The discovery of divergent modes of Hh secretion and the “2nd cleavage” open novel avenues for Hh research by offering an alternative, and very direct, line of attack in the treatment of Hh-related cancer.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10288400 |
| Date | January 2012 |
| Creators | Daniele, Joseph |
| Contributors | Kunes, Samuel M. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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