Vertebrate SUFU plays a critical role in the negative regulation of Hh signaling in the absence of ligand. Disruption of SUFU causes constitutive activation of the pathway, and is associated with the development of medulloblastoma in humans (Cooper et al, 2005; Svard et al, 2006; Taylor et al, 2002; Pastorino et al, 2009). SUFU binds directly to all three GLI proteins (Pearse et al, 1999; Stone et al, 1999; Jia et al, 2009; Svard et al, 2006). Formation of a SUFU:GLI complex is required for the processing of GLI3 to the GLI3R repressor form, and the processing depends on transit through the primary cilia (Kise et al, 2009; Humke et al, 2010; Huangfu and Anderson, 2005). Despite this, primary cilia are not required for SUFU to inhibit GLI activity; SUFU may also serve in a cilia-independent manner to sequester the full-length protein in the cytoplasm in the absence of Hh signal (Chen et al, 2009; Humke et al, 2010; Jia et al, 2009; Tukachinsky et al, 2010). After processing, GLI3R dissociates from SUFU and its activity is SUFU-independent (Humke et al, 2010; Tukachinsky et al, 2010). Nuclear SUFU may also play a direct role as a transcriptional co-repressor through interaction with the N-terminal DNA-binding domain of GLI proteins, though this remains to be fully elaborated (Monnier et al, 1998; Pearse et al, 1999; Cheng and Bishop, 2002; Paces-Fessy et al, 2004; Dunaeva et al, 2003; Szczepny et al, 2014).

ULK3 is a serine-threonine kinase that was identified as a positive regulator of Hh signaling that regulates GLI activity by phosphorylating the full-length form (Maloverjan et al, 2010a). In the absence of Hh ligand, ULK3 forms a complex with SUFU that restricts its kinase activity (Maloverjan et al, 2010b). Upon Hh stimulation, the ULK3:SUFU complex dissociates, allowing ULK3 to phosphorylate the full-length GLI proteins and promoting their activation and nuclear localization (Maloverjan et al, 2010a; Maloverjan et al, 2010b). ULK3 is related by sequence to the vertebrate kinase STK36, homologue to Drosophila Fused (Fu). While Fu plays a critical role in propagating Hh signal and is part of the Hedgehog signaling complex (HSC), STK36 is not required for Hh signaling in vertebrate cells but instead contributes to the formation of motile cilia (Wilson et al, 2009; reviewed in Briscoe and Therond, 2013; Maloverjan and Piirsoo, 2012).

SPOP:CUL3:RBX1-mediated ubiquitination of the transcriptionally active GLI proteins attenuates Hh-dependent signaling by promoting their degradation by the proteasome (Zhang et al, 2009; Chen et al, 2009; Humke et al, 2010; Tukachinsky et al, 2010; Wen et al, 2010).

The transcriptional activity of full-length activated Ci/GLI proteins is restricted by their rapid ubiquitin-mediated degradation after initiation of Hh signaling (Ohlmeyer et al, 1998; Humke et al, 2010; Tukachinsky et al, 2010; Wen et al, 2010). Ubiquitination of Ci, GLI2 and GLI3 is mediated by the E3 ligase complex SPOP:CUL3:RBX1, which ubiquitinates the transcription factors in a Hh-dependent manner (Zhang et al, 2006; Kent et al, 2006; Zhang et al, 2009; Chen et al, 2009).

IQCE and EFCAB7 are ciliary proteins that are required to restrict the EVC2:EVC complex to the 'EVC region' at the base of the cilium, just distal to the transition zone (Pusapati et al, 2014). EVC2 and EVC are transmembrane proteins that form a ciliary-localized complex that is a positive regulator of Hh signal transduction. The EVC2:EVC complex appears to act downstream of both SMO ciliary localization and its activation by CSNK1A1 and ADRBK1, and is required for the dissociation of the GLI:SUFU complex at the ciliary tip, although the mechanism for this is not known (Blair et al, 2011; Dorn et al, 2012; Capparos-Martin et al, 2013; Pusapati et al, 2014). EVC2 interacts with the IQCE:EFCAB7 subcomplex through the so called 'W-peptide', a stretch of amino acids in the intracellular tail that is deleted in the ciliopathy Weyers Acrofacial Dysostosis. Deletion of the W-peptide results in mislocalization of EVC2 throughout the length of the cilium, rather than being concentrated in the 'EVC zone' (Pusapati et al, 2014; Dorn et al, 2012; Capparos-Martin et al, 2011). EVC2:EVC localization to the EVC region, mediated by the IQCE:EFCAB7 complex and the W-peptide, is required for the Hh-dependent activation of full-length GLI2, but does not appear to critical for the regulation of GLI3R levels, suggesting a bifurcation of the pathway (Pusapati et al, 2014).

EVC2 and EVC are components of a complex that localizes to the base of the cilium in a so-called EvC zone just distal to the transition zone. Mutations in the genes for EVC2 and EVC are associated with the ciliopathy Ellis van Creveld syndrome and result in an abrogated response to stimulation by Hh, making EVC2 and EVC positive regulators of Hh signaling (Blair et al, 2011; Dorn et al, 2012; Caparros-Martin et al, 2013). The EVC2:EVC complex interacts with SMO in the cilium after Hh stimulation and restricts SMO localization to the EvC zone (Dorn et al, 2012; Yang et al, 2012; Caparros-Martin et al, 2013). Disruption of the EVC2:EVC complex does not interfere with SMO ciliary localization or its activation by CSNK1A1 and ADRBK1, but prevents the Hh-dependent localization of the GLI transcription factors to the tip of the cilium and abrogates the dissociation of the GLI:SUFU complex (Dorn et al, 2012; Yang et al, 2012; Caparros-Martin et al, 2013). These events are required for the activation of the GLI transcription factors in response to ligand stimulation. Localization of the EVC2:EVC complex to the EVC zone depends on an interaction between the EVC2 W peptide (a stretch of 43 amino-acids in the C-terminal tail that is missing in a disease associated EVC2-variant), and the IQCE:EFCAB7 complex. Abrogation of this interaction causes the EVC2:EVC complex to localize along the length of the cilium and disrupts production and nuclear translocation of the full length GLI2 transcriptional activator (Pusapati et al, 2014). How the Hh signal is transmitted from the SMO:EVC2:EVC complex to downstream components is not known.

Suppressor of fused homolog