Multisite protein kinase A and glycogen synthase kinase 3β phosphorylation leads to Gli3 ubiquitination by SCFβTrCP

Gli3 is a zinc finger transcription factor proteolytically processed into a truncated repressor lacking C-terminal activation domains. Gli3 processing is stimulated by protein kinase A (PKA) and inhibited by Hedgehog signaling, a major signaling pathway in vertebrate development and disease. We show here that multisite glycogen synthase kinase 3 beta (GSK3 beta) phosphorylation and ubiquitination by SCF beta TrCP are required for Gli3 processing. We identified multiple beta TrCP-binding sites related to the DSGX(2-4)S motif in Gli3, which are intertwined with PKA and GSK3 beta sites, and SCF beta TrCP target lysines that are essential for processing. Our results support a simple model whereby PYA triggers a cascade of Gli3 phosphorylation by GSK3 beta and CK1 that leads to direct beta TrCP binding and ubiquitination by SCF beta TrCP. Binding of beta TrCP to Gli3 N- and C-terminal domains lacking DSGX(2-4)S-related motifs was also observed, which could reflect indirect interaction via other components of Hedgehog signaling, such as the tumor suppressor Sufu. Gli3 therefore joins a small set of transcription factors whose processing is regulated by the ubiquitin-proteasome pathway. Our study sheds light on the role of PFA phosphorylation in Gli3 processing and will help to analyze how dose-dependent tuning of Gli3 processing is achieved by Hedgehog signaling.