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

Abstract

Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. 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 βTrCP/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 βTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank the members of the Genentech Hedgehog team for stimulating discussions; Curis, Inc., (Cambridge, MA) for HhAntag; Mark Merchant and Setsu Endoh-Yamagami for isolating MEFs; Derek Marshall and Tracy Tang for certain TaqMan primer/probes; Vivian Barry for the Gli antigen constructs; Cecilia Brown, Manda Wong, and Hong Li for antigen expression and purification; James Ernst and Hong Li for octylated Shh; Joe Beirao at Josman Labs (Napa, CA) for rabbit immunization; Antibody Solutions (Mountain View, CA) for serum titer determinations; and Kurt Schroeder for affinity purification of the MAbs and Smo pAb. We are grateful for Erik Huntzicker's and Andy Peterson's helpful comments on the manuscript.