Kinetics of hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation.
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 betaTrCP/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 betaTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.
Genentech, South San Francisco, CA 94080, USA.
Xiaohui
X
Wen
Cary K
CK
Lai
Marie
M
Evangelista
Jo-Anne
JA
Hongo
Frederic J
FJ
de Sauvage
Suzie J
SJ
Scales
D000818:Animals; D002460:Cell Line; D002923:Cilia / Q000378:metabolism* / Q000648:ultrastructure; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D000072079:Patched Receptors; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D000072086:Smoothened Receptor; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3
C000618551:Gli2 protein, mouse; C465571:Gli3 protein, mouse; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D000072079:Patched Receptors; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3; D017868:Cyclic AMP-Dependent Protein Kinases; D046988:Proteasome Endopeptidase Complex