Mechanisms regulating protein stability of Gli

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DESCRIPTION (provided by applicant): Inappropriate Sonic hedgehog (Shh) target gene induction has been implicated in many human cancers, including skin Basal cell carcinomas (BCCs). Previous studies have shown that regulation of Shh signal reception in skin keratinocytes occurs in part by regulating Gli transcription factor protein stability. Preliminary data has demonstrated two regions of Gli that mediate destruction. C-terminal sequences contain a ubiquitin-proteasome destruction signal or degron that is recognized by the adapter protein, ¿TrCP, a ubiquitin ligase. However, the identity of the putative N-terminal degron and the complex that binds to it remains unknown. The goal of this proposal is to identify the N-terminal sequences and trans-acting factors that bind to it to mediate Gli protein degradation. We aim to: 1) Determine the minimal sequences that define the degron and are sufficient to confer protein instability to chimeric proteins via deletion mutant analysis and degradation assays. We will assess how known regulators of Shh signaling affect both the N and C terminal degrons; 2) Identify Gli1 N-terminal associated proteins via protein pull-down assays and mass spectrometry technology. We will characterize Gli1 N-terminal associated proteins in Gli1 protein stability and ubiquitination assays using loss and gain of function analyses.