Hedgehog signal transduction: mechanism of Ci/Gli transcriptional effector activation

Project Summary This project aims to understand the basic molecular mechanisms of Hedgehog (Hh) signal transduction in Drosophila. Hh proteins are prominent members of the limited set of extracellular signaling molecules responsible for directing development and regulating stem cell activity in Drosophila and in humans. Consequently, genetic alterations to Hh signaling underlie many developmental disorders and many types of cancer. Pioneering studies in Drosophila identified the central Hh signaling pathway components, which were later found to be highly conserved in mammals. Those advances led to much improved diagnosis of several human syndromes and diseases, and to the development of very promising anti-cancer drugs that target Smoothened (Smo), a key membrane protein that initiates Hh signal transduction. The Hh pathway culminates in altering patterns of gene transcription by modifying the activity of the conserved transcription factors, Ci (in flies) and Gli1-3 (in mammals). Deciphering the mechanisms that connect Smo to Ci/Gli activation is critical to understand Hh signaling and for the development of drugs that directly target Ci/Gli activity. Such drugs would potentially counter or restore pathway activation perturbed by any type of mutation. Hh signaling is extremely sensitive to the stoichiometry of signal transduction components and must therefore be studied under normal physiological conditions. Drosophila offers the most rapid and incisive molecular genetic approaches to accomplish this. New insights can then be applied rapidly to Hh signaling in humans and other mammals because the components and mechanisms involved are highly conserved. Hh regulates Ci in two ways. Hh blocks proteolytic processing of full-length Ci (Ci-155) to a transcriptional repressor and it activates Ci-155, opposing inhibition by a direct binding partner, Suppressor of fused (Su(fu)). The mechanism of Ci or Gli activation is largely unknown, but the protein kinase, Fused (Fu), has a central role in Drosophila. We aim to understand how Su(fu) restrains Ci-155 activity and how Fu kinase overcomes this inhibition. Our studies will use extensive resources generated by prior studies: a large set of CRISPR-derived designer ci alleles, stocks that additionally permit simultaneous genetic alteration of one or more interacting signal transduction proteins and knowledge of Fu target sites of uncertain functional importance. We expect to define relevant Fu sites, investigate indications that Hh promotes a transition from an inactive to an active Ci-Su(fu) complex, and explore whether these regulatory transitions can be reproduced by relevant domains of mammalian proteins in Drosophila.