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 3␤ (GSK3␤) phosphorylation and ubiquitination by SCF ␤TrCP are required for Gli3 processing. We identified multiple ␤TrCP-binding sites related to the DSGX 2-4 S motif in Gli3, which are intertwined with PKA and GSK3␤ sites, and SCF ␤TrCP target lysines that are essential for processing. Our results support a simple model whereby PKA triggers a cascade of Gli3 phosphorylation by GSK3␤ and CK1 that leads to direct ␤TrCP binding and ubiquitination by SCF ␤TrCP . Binding of ␤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 PKA phosphorylation in Gli3 processing and will help to analyze how dose-dependent tuning of Gli3 processing is achieved by Hedgehog signaling.

Dual Phosphorylation of Suppressor of Fused (Sufu) by PKA and GSK3β Regulates Its Stability and Localization in the Primary Cilium

“…1G), whereas S342D/S346D showed weak activating and S301A/T305A showed little effect. The stabilizing effect of PKA on Sufu is consistent with its negative regulation of Shh signaling (10,33).…”

“…Transcription of Shh target genes is controlled by three Gli transcription factors of the Kruppel zinc finger DNA-binding protein family (8), among which Gli2 and Gli3 exist in two forms as follows: the full-length transcription activators and the truncated N-terminal fragments generated by a partial proteolysis of the C termini (5,9,10). Nascent Gli proteins are labile, requiring the binding of Sufu for stable expression (11)(12)(13).…”

Hedgehog-induced phosphorylation by CK1 sustains the activity of Ci/Gli activator

“…In the absence of Hh, CK1 phosphorylates Ci/Gli after PKA-primed phosphorylation, which is essential for the production of Ci R /Gli R (34)(35)(36)(37)(38); however, in the presence of Hh, CK1 phosphorylates Smo and likely Fu, to activate the Hh pathway (15,30,(39)(40)(41). Here we uncover an unanticipated positive role of CK1 in the regulation of Ci A downstream of Smo and Fu.…”

Evidence for the Direct Involvement of the Proteasome in the Proteolytic Processing of the Aspergillus nidulans Zinc Finger Transcription Factor PacC

“…8), as expected if ubiquitination were coupled to phosphorylation. Such a sequential mechanism has been reported for NF-B p105 (6) but, most notably, for the processing of Ci/Gli transcription factors, where a phosphorylation cascade promotes ␤TrCP recruitment to mediate ubiquitination and subsequent proteolytic processing (5,(57)(58)(59)(60). The remarkable similarities with the zinc finger DNA binding domains and the mechanisms of regulation of Ci/Gli have been underlined previously (20).…”