The activity of the Gli proteins is regulated by processing and degradation, both of which are mediated by ubiquitination. Whereas the degradation of Gli proteins reduces the amount of the protein, the processing generates a cleaved Gli form which acts as a negative regulator (Jiang, 2006). The three Gli proteins exhibit different activities on the target promoter sequence. Gli1 has the highest promoter-inducing activity, Gli2 exhibits moderate activity, and Gli3 is mainly inhibitory. These differences are partly explained by the presence of the cleaved form of the Gli proteins. Gli3 is largely found in a cleaved form in cells, whereas only a limited subset of Gli2 is subjected to cleavage (Pan et al., 2006). The activity of the Gli transcription factor is correlated with the ratio of the cleavage and the amount of the inhibitory form existing in cells. Gli1 is not cleaved to form the inhibitory molecule (Dai et al., 1999; Kaesler et al., 2000). The processing step was originally identified for the Drosophila Gli homolog Ci, which is processed by the Slimb ubiquitin ligase (Aza-Blanc et al., 1997; Jiang and Struhl, 1998). The 190 kDa Gli3-190 is cleaved and forms an 83 kDa Gli-83 upon co-expression with β-Trcp, a mammalian homolog of Slimb (Pan et al., 2006). Gli3 is phosphorylated by PKA, CK1, and GSK-3 (Tempe et al., 2006; Wang and Li, 2006), and β-Trcp only interacts with the phosphorylated form of Gli3. Gli3 is subjected to ubiquitination, presumably by the SCFβ-Trcp ubiquitin ligase complex, and is processed in the proteasome. Inhibition of proteasome activity suppresses the formation of Gli-83 form (Figure 3B) (Wang and Li, 2006). A similar processing mechanism is also present for Gli2, which generates Gli2-78 from Gli2-185 (Figure 3B). However, the efficiency of Gli2 processing is lower in comparison with Gli3, suggesting that Gli3 is the preferential target for the SCFβ-Trcp complex. Ubiquitination of Gli2 is also mediated by SCFβ-Trcp (Figure 3B) (Bhatia et al., 2006), although how the same ligase regulates the processing and degradation mechanisms is unknown. The processing mechanisms of Gli1 have not yet been reported; however, Gli1 is also subjected to ubiquitination and subsequent degradation. Gli1 has two degradation domains: the N-terminal degron DN and the C-terminal degron DC (Huntzicker et al., 2006). The degron DC is conserved in the Gli2 degradation domain and interacts with β-Trcp (Figure 3B). The degron DN is also highly conserved among the three Gli proteins. The degron DN induces the degradation of Gli1 independently of the DC. Complete stabilization of the Gli1 is achieved by mutations in both the DN and DC regions. Constitutive expression of Gli1 in keratinocytes is sufficient to induce BCC, albeit at a later stage, at up to 6 months of age (Huntzicker et al., 2006). However, the expression of the most stable form of the deletion mutant (the degron DN and DC double mutant) in the skin of mice leads to the formation of BCCs at the perinatal stage and causes death. The ubiquitin ligase responsible for degron DN-mediated degradation is still uncharacterized. Another ubiquitin ligase which targets Gli for degradation, HIB/SPOP, has recently been identified. HIB/SPOP is a BTB domain protein, and is present in the Cullin3 complex. HIB/SPOP recognizes the serine/threonine-rich sequence in the N- and C- terminal regions of the Ci protein in Drosophila (Zhang et al., 2009). The expression of HIB/SPOP is upregulated by the HH pathway, and the HIB/SPOP-mediated degradation of Gli1 appears to constitute a negative feedback loop (Figure 3A). Therefore, the Gli1 protein is regulated by the two complex types of ubiquitin ligases, SCF β-Trcp and Cul3-HIB/SPOP. Moreover, the HECT-type ubiquitin ligase Itch targets Gli1 for ubiquitination-dependent degradation (Figure 3B) (Di Marcotullio et al., 2006). In this case, Numb serves as an adaptor between Itch and Gli1 and induces the degradation of Gli1. Therefore, Numb functions as a negative regulator of HH signaling.