Biochemical Journal

Research article

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

Mélanie PACES-FESSY, Dominique BOUCHER, Emile PETIT, Sandrine PAUTE-BRIAND, Marie-Françoise BLANCHET-TOURNIER

Abstract

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.

  • Galectin3
  • Gli
  • SAP18
  • Suppressor of fused (Sufu)

Footnotes

  • 1 Present address: Laboratoire de Génétique et Biologie Cellulaire, Bâtiment BUFFON, 4ème étage, Université de Versailles-Saint-Quentin-en-Yvelines 45, avenue des Etats-Unis, 78035 Versailles, France.

  • 2 Present address: Centre de Recherches sur la Cognition Animale, CNRS UMR 5169, Université Paul Sabatier (Toulouse 3), 118 Route de Narbonne, Bât. 4R3, 31062 Toulouse cedex, France.

  • Abbreviations used: AD, activation domain; CBF1, CCAAT-binding factor 1; Ci, Cubitus interruptus; CBP, cAMP-response-element-binding protein; dpc, day post-coïtum; Sufu, Suppressor of fused; dSufu, Drosophila Sufu; mSufu, mouse Sufu; GFP, green fluorescent protein; GSK, glycogen synthase kinase; GST, glutathione S-transferase; Hh, Hedgehog; HMGiy, high mobility group; hnRNP, heterogeneous nuclear ribonucleoprotein; LMB, leptomycin B; MALDI, matrix-assisted laser-desorption ionization; NES, nuclear export signal; pCIP, p300/CBP-co-integrator protein; PIAS1, protein inhibitor of activated STAT1; PKA, protein kinase A; SAP18, Sin3-associated polypeptide 18; STAT, signal transduction and activators of transcription; SUMO, small ubiquitin-related modifier; X-Gal, 5-bromo-4-chloroindol-3-yl β-d-galactopyranoside.