Tempé Denis, Casas Mariana, Karaz Sonia, Blanchet-Tournier Marie-Françoise, Concordet Jean-Paul
Département Génétique et Développement, Institut Cochin, 24 rue du Faubourg St-Jacques, 75014 Paris, France.
Mol Cell Biol. 2006 Jun;26(11):4316-26. doi: 10.1128/MCB.02183-05.
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.
Gli3是一种锌指转录因子，经蛋白水解处理后成为一种缺乏C端激活结构域的截短型阻遏物。Gli3的处理过程受蛋白激酶A（PKA）刺激，并受Hedgehog信号通路抑制，Hedgehog信号通路是脊椎动物发育和疾病中的主要信号通路。我们在此表明，Gli3的处理需要多位点糖原合酶激酶3β（GSK3β）磷酸化以及SCFβTrCP介导的泛素化。我们在Gli3中鉴定出多个与DSGX2 - 4S基序相关的βTrCP结合位点，这些位点与PKA和GSK3β位点相互交织，且SCFβTrCP靶向的赖氨酸对于处理过程至关重要。我们的结果支持一个简单模型，即PKA触发GSK3β和CK1对Gli3的磷酸化级联反应，导致SCFβTrCP直接结合并使其泛素化。还观察到βTrCP与缺乏DSGX2 - 4S相关基序的Gli3 N端和C端结构域结合，这可能反映了通过Hedgehog信号通路的其他成分（如肿瘤抑制因子Sufu）进行的间接相互作用。因此，Gli3加入了一小类其处理过程受泛素 - 蛋白酶体途径调控的转录因子。我们的研究揭示了PKA磷酸化在Gli3处理中的作用，并将有助于分析Hedgehog信号通路如何实现对Gli3处理的剂量依赖性调节。
