Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP.
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 3beta (GSK3beta) phosphorylation and ubiquitination by SCFbetaTrCP are required for Gli3 processing. We identified multiple betaTrCP-binding sites related to the DSGX2-4S motif in Gli3, which are intertwined with PKA and GSK3beta sites, and SCFbetaTrCP target lysines that are essential for processing. Our results support a simple model whereby PKA triggers a cascade of Gli3 phosphorylation by GSK3beta and CK1 that leads to direct betaTrCP binding and ubiquitination by SCFbetaTrCP. Binding of betaTrCP to Gli3 N- and C-terminal domains lacking DSGX2-4S-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.
Tempé
Denis
D
Département Génétique et Développement, Institut Cochin, 24 rue du Faubourg St-Jacques, 75014 Paris, France.
Casas
Mariana
M
Karaz
Sonia
S
Blanchet-Tournier
Marie-Françoise
MF
Concordet
Jean-Paul
JP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D016415:Sequence Alignment; D025801:Ubiquitin / Q000378:metabolism*; D000074290:Zinc Finger Protein Gli3
C065683:GLI3 protein, human; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D025801:Ubiquitin; D000074290:Zinc Finger Protein Gli3; D044843:SKP Cullin F-Box Protein Ligases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D008239:Lysine