Ubiquitin ligase regulation of Gli transcription factors

Inappropriate spatial or temporal activation of the Sonic Hedgehog (Shh) signaling pathway in the skin results in the development of basal cell carcinomas (BCCs). Normal Shh activation of Gli1 and Gli2 transcription factors in the skin is restricted to the hair follicle. We have identified two protein sequences or degrons, DN and DC, that independently regulate Gli1 protein degradation by the proteasome in cultured cells. We also determined these degrons to regulate Gli protein stability in the skin as overexpression of Gli1 DN and DC mutants in the basal layer of the skin accelerated BCC tumor development. DC is a hexapeptide motif, DSGVEM, that resembles the canonical binding site for βTrCP, a substrate adaptor for a Cullin1 ubiquitin ligase (SCFβTrcP). SCF βTrCP recognizes DC in Gli1 to promote complete proteasome-dependent proteolysis of Gli1. SCFβTrCP was also found to promote Gli2 and Gli3 proteolytic processing into repressor forms and its activity was inhibited by Shh signaling (Pan et al. 2006, Wang & Li 2006, Huangfu & Anderson). We have investigated the mechanism underlying the D N destruction signal. We find that speckle-type POZ protein (SPOP), a substrate adaptor for a Cullin3 (Cul3) ubiquitin ligase, recognizes Gli1, Gli2, and Gli3 proteins and targets them for proteasome-dependent degradation. Overexpressed SPOP colocalized with Gli activator proteins in Shh-stimulated cells and attenuated Shh signaling in Gli-luciferase and endogenous Shh target gene induction assays. A dominant negative form of SPOP (SPOP-DN) stabilized Gli1 and Gli2 protein and enhanced their transcriptional activity. SPOP-DN also blocked constitutive ubiquitinylation of Gli2 in HEK293T cells. SPOP regulation of Gli1 protein stability and subsequent transcriptional activity was mediated through DN, however, SPOP did not directly recognize Gli1DN. Unlike its fly homolog (HIB/Rdx), SPOP is not a target of the Hh pathway, as SPOP transcription was not induced in Shh-stimulated mouse embryonic fibroblasts (MEFs) or in BCC tissue samples. We also made the surprising discovery that vertebrate SPOPs, SPOP1 and SPOP2, are required for maximal Shh signaling in various MEFs. We suspect that SPOP proteins may be involved in other cellular processes that may in turn act to regulate the Shh pathway. We conclude that SPOP/Cul3 and SCFβTrCP represent highly conserved dual degradation pathways for Ci/Gli proteins. These pathways are distinctly regulated by Shh to prevent ectopic Shh target gene induction in the absence of Shh stimulation and to attenuate Shh signaling after pathway activation.