Kinetics of Hedgehog-Dependent Full-Length Gli3 Accumulation in Primary Cilia and Subsequent Degradation

Abstract

Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and ␤TrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or ␤TrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.

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“…Despite previous in vitro data suggesting an important role of Spop in Gli2 degradation (18)(19)(20)(21)23), the level of Gli2 was not significantly changed in Spop mutants (Fig. 4C).…”

“…Previous in vitro studies found that Spop inhibited Hh signaling by targeting Gli2 and Gli3FL for degradation (18)(19)(20)(21)23). To test whether Ihh signaling was affected by the loss of Spop, we examined the expression of Patched 1 (Ptch1), a direct transcriptional target of Ihh, in E13.5 forelimbs.…”

“…Speckle-type POZ protein (Spop) is the substrate-recognition subunit of a Cullin-Ring E3 ubiquitin ligase that targets mammalian Gli2 and the full-length form of Gli3 (Gli3FL) for ubiquitination and degradation in vitro (18)(19)(20)(21). The Spop homolog in Drosophila, known as hib or rdx, also mediates the degradation of Ci, the sole Gli family member in flies, and inhibits Hh signaling (22,23).…”

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“…It is the relative efficiency with which these two Gli forms are specifically proteolyzed that distinguishes the inactive versus the active hedgehog signaling state. In the absence of activated Smo, Gli2 and 3 proteins traffic into primary cilium where they are modified into repressor forms [41]. This process is initiated by a series of sequential phosphorylations, initiated by protein kinase A and then followed by glycogen synthase kinase-3-β and casein kinase 1.…”

“…For the target cell, hedgehog signaling can be facilitated by the presence of heparin proteoglycans and lower affinity hedgehog coreceptor proteins that include CDON and BOC [49]. Further downstream, integration of vertebrate hedgehog signaling into the primary cilium means that signal processing requires the activities of numerous ciliar transport proteins to shuttle Gli proteins into and out of the cilium [41,50,51]. Genetic ablation of individual ciliar transport proteins in mice confers phenotypes that are reiterative of mutations in the primary hedgehog regulatory genes.…”