Quelques aspects des interactions hormonales entre la mue et la vitellogenèse chez le Crustacé Amphipode Orchestia gammarellus (Pallas)

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

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)(-)(4)S 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)(-)(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

The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins.

Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein. We identified several mSufu variants of which some were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. We confirmed the mSufu-SAP18 and mSufu-Galectin3 interactions by independent biochemical assays. Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus