RECONSTITUTION OF HEDGEHOG SIGNALING COMPLEX

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Mutations in components of the Hedgehog (Hh) signal transduction pathway underlie a number of human developmental disorders, and contribute to a diverse array of tumors. Gorlin's syndrome is one example of the role this pathway plays in both development and cancer. Besides numerous developmental abnormalities, individuals afflicted with this disorder have an inherited predisposition to basal cell carcinoma as well as to a variety of primitive neuroectodermal tumors, such as medulloblastoma. Similar components are also found mutated in sporadic forms of these same malignancies, or highly amplified in glioblastoma. Our long-term goals are to elucidate how this signal transduction pathway is usurped in these different human pathologies. Hedgehog signal transduction occurs through a large multi-component microtubule-bound protein complex, that when disrupted blocks signaling. This Hedgehog signaling complex (HSC) is an approximately 1000 kDa multi-protein complex with three known members, the protein kinase Fused (Fu), the transcription factor Cubitus Interuptus (Ci), and the kinesin-like protein Costal2 (Cos2). In addition to Fu, Ci, and Cos2, we hypothesize that the HSC contains a number of as yet unknown, but functionally important protein components. The goals of this project are to identify these components, and evaluate their role in HSC function and organization. The specific aims of our studies are: 1) Develop an in vitro reconstitution system for members of the HSC using a baculovirus expression system, this will allow us to isolate modified forms of the various HSC components in large amounts for various in vitro studies; 2) Identify additional members of a Hh dependent signaling complex. This will be done through a combination of both affinity and conventional chromatography, as well as using a candidate gene approach. Identifying these other components will provide insight into the role this large protein machine plays in Hh signal transduction. It will also provide the framework necessary to identify the human orthologs of the HSC, and elucidate their role in oncogenesis (in future work).