Multisite Protein Kinase A and Glycogen Synthase Kinase 3β Phosphorylation Leads to Gli3 Ubiquitination by SCF^βTrCP

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.

D.T. was supported by doctoral fellowships from MNRT and ARC, and M.C. was supported by a doctoral fellowship from the Ministerio de Planificacion Nacional (Chile). This work was supported by INSERM and ARC.

We are grateful to the Benarous laboratory for βTrCP expression plasmids and discussion. B. Vogelstein, H. Sasaki, D. Bohmann, M. Raymonjean, M. Pap, G. M. Cooper, M. J. Birnbaum, and K. Hattori kindly provided plasmids. We thank F. Letourneur and his colleagues at the DNA Sequencing Facility of Institut Cochin.