Multisite Protein Kinase A and Glycogen Synthase Kinase 3β Phosphorylation Leads to Gli3 Ubiquitination by SCFβTrCP

Abstract

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 3β (GSK3β) phosphorylation and ubiquitination by SCF^βTrCP are required for Gli3 processing. We identified multiple βTrCP-binding sites related to the DSGX_2-4S motif in Gli3, which are intertwined with PKA and GSK3β sites, and SCF^βTrCP target lysines that are essential for processing. Our results support a simple model whereby PKA triggers a cascade of Gli3 phosphorylation by GSK3β and CK1 that leads to direct βTrCP binding and ubiquitination by SCF^βTrCP. Binding of βTrCP to Gli3 N- and C-terminal domains lacking DSGX_2-4S-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 PKA phosphorylation in Gli3 processing and will help to analyze how dose-dependent tuning of Gli3 processing is achieved by Hedgehog signaling.