Evidence for a novel feedback loop in the Hedgehog pathway involving the seven transmembranedomain protein Smoothened and the kinase Fused.

International audienceHedgehog (HH) is a major secreted morphogen involved in development, stem cell maintenance and oncogenesis[1, 2].In Drosophila wing imaginal discs, HH produced in the posterior compartment diffuses into the anterior compartment to control target gene transcription via the transcription factor Cubitus interruptus (CI). The first steps in reception and transduction of the HH signal are mediated by its receptor Patched (PTC)[3] and the seven-transmembranedomain protein Smoothened (SMO) [4, 5]. PTC and HH control SMOby regulating its stability, trafficking and phosphorylation (for review see [6]). SMO interacts directly with the Ser-Thr protein kinase Fused (FU) and the kinesin-related protein Costal2 (COS2), which interact with each other and with CI in an intracellular “Hedgehog transducing complex”[7-9].We show here that HH induces FUtargeting to the plasma membrane in a SMO-dependent fashion and that,reciprocally,FUcontrols SMO stability and phosphorylation. FUanchorage to the membrane is sufficient to make it a potent SMO-dependent, PTC-resistant, activator of the pathway. These findings reveal a novel positive-feedback loop in HH transduction and are consistent with a model in which FU and SMO, by mutually enhancing each other's activities, sustain high levels of signalingand render the pathway robust to PTClevel fluctuations

Hedgehog signal transduction proteins: contacts of the Fused kinase and Ci transcription factor with the Kinesin-related protein Costal2

BACKGROUND: Hedgehog signaling proteins play important roles in development by controlling growth and patterning in various animals including Drosophila and mammals. Hedgehog signaling triggers changes in responsive cells through a novel transduction mechanism that ultimately controls the transcription of specific target genes via the activity of zinc finger transcription factors of the Cubitus interruptus /GLI family. In flies, key Hedgehog signal transduction components have been identified including the kinesin-related protein Costal2, the serinethreonine kinase Fused, and the PEST-containing protein Suppressor of Fused. These proteins control Cubitus interruptus cleavage, nucleo-cytoplasmic localization and activation. In fly embryos, Costal2, Fused, Suppressor of Fused and Cubitus interruptus are associated in at least one cytoplasmic complex, which interacts with the microtubules in a Hedgehog-dependent manner. RESULTS: Here we identified and mapped direct interactions between Cos2, Fu, and Ci using an in vitro affinity assay and the yeast two-hybrid system. CONCLUSIONS: Our results provide new insights into the possible mechanism of the cytosolic steps of Hedgehog transduction

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.Fil: Bruzzone, Lucia. Centre National de la Recherche Scientifique; Francia. Universite de Paris; FranciaFil: Argüelles, Camilla. Centre National de la Recherche Scientifique; Francia. Universite de Paris; FranciaFil: Sanial, Matthieu. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Miled, Samia. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Alvisi, Giorgia. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Gonçalves Antunes, Marina. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Qasrawi, Fairouz. Universite de Paris; Francia. Centre National de la Recherche Scientifique; FranciaFil: Holmgren, Robert A.. Northwestern University; Estados UnidosFil: Smibert, Craig A. University of Toronto; CanadáFil: Lipshitz, Howard D.. University of Toronto; CanadáFil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Plessis, Anne. Centre National de la Recherche Scientifique; Francia. Universite de Paris; FranciaFil: Bécam, Isabelle. Centre National de la Recherche Scientifique; Francia. Universite de Paris; Franci

CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder with unclear underlying mechanisms. Here, the authors unravel the contribution of a stress-responsive pathway to RSTS where impaired HSF2 acetylation, due to RSTS-associated CBP/EP300 mutations, alters the expression of neurodevelopmental players, in keeping with hallmarks of cell-cell adhesion defects.Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.</p

Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases.

International audienceThe conserved Hedgehog (HH) signals control animal development, adult stem cell maintenance and oncogenesis. In Drosophila, the HH co-receptor Patched (PTC) controls both HH gradient formation and signalling. PTC is post-translationally downregulated by HH, which promotes its endocytosis and destabilization, but the mechanisms of PTC trafficking and its importance in the control of PTC remain to be understood. PTC interacts with E3 Ubiquitin (UB)-ligases of the C2-WW-HECT family; two of them-SMURF and NEDD4-are known to regulate its levels. We demonstrate that mutation of the PTC PY motif, which mediates binding of C2-WW-HECT family members, inhibits its internalization but not its autonomous and non-autonomous signalling activities. In addition, we show that the two related UB-C2-WW-HECT ligases NEDD4 and SU(DX) regulate PTC trafficking and finely tune its accumulation through partially redundant but distinct functions. While both NEDD4 and SU(DX) promote PTC endocytosis, only SU(DX) is able to induce its lysosomal targeting and degradation. In conclusion, PTC trafficking and homeostasis are tightly regulated by a family of UB-ligases

Characterization of the Drosophila myeloid leukemia factor.

International audienceIn human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, dMLF physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. Here, we show that dmlf is widely and dynamically expressed during fly development. We isolated and analyzed the first dmlf mutants: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf(-)- adults display weak phenotypes. We monitored dMLF subcellular localization in the fly and cultured cells. We were able to show that, although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, we demonstrate that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model

Biophysical characterisation of the novel zinc binding property in Suppressor of Fused

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The last 59 amino acids of Smoothened cytoplasmic tail directly bind the protein kinase Fused and negatively regulate the Hedgehog pathway.

International audienceThe Hedgehog (HH) signaling pathway is crucial for the development of many organisms and its inappropriate activation is involved in numerous cancers. HH signal controls the traffic and activity of the seven-pass transmembrane protein Smoothened (SMO), leading to the transcriptional regulation of HH-responsive genes. In Drosophila, the intracellular transduction events following SMO activation depend on cytoplasmic multimeric complexes that include the Fused (FU) protein kinase. Here we show that the regulatory domain of FU physically interacts with the last 52 amino acids of SMO and that the two proteins colocalize in vivo to vesicles. The deletion of this region of SMO leads to a constitutive activation of SMO, promoting the ectopic transcription of HH target genes. This activation is partially dependent of FU activity. Thus, we identify a novel link between SMO and the cytoplasmic complex(es) and reveal a negative role of the SMO C-terminal region that interacts with FU. We propose that FU could act as a switch, activator in presence of HH signal or inhibitor in absence of HH

Costal2 Functions as a Kinesin-like Protein in the Hedgehog Signal Transduction Pathway

SummaryThe Hedgehog (Hh) signaling pathway initiates an evolutionarily conserved developmental program required for the proper patterning of many tissues [1]. Although Costal2 (Cos2) is a requisite component of the Hh pathway, its mechanistic role is not well understood. Because of its primary sequence, Cos2 was initially predicted to function as a kinesin-like protein [2]. However, evidence showing that Cos2 function might require kinesin-like properties has been lacking [2–6]. Thus, the prevailing dogma in the field is that Cos2 functions solely as a scaffolding protein [7, 8]. Here, we show that Cos2 motility is required for its biological function and that this motility may be Hh regulated. We show that Cos2 motility requires an active motor domain, ATP, and microtubules. Additionally, Cos2 recruits and transports other components of the Hh signaling pathway, including the transcription factor Cubitus interruptus (Ci). Drosophila expressing cos2 mutations that encode proteins that lack motility are attenuated in their ability to regulate Ci activity and exhibit phenotypes consistent with attenuated Cos2 function [9]. Combined, these results demonstrate that Cos2 motility plays an important role in its function, regulating the amounts and activity of Ci that ultimately interpret the level of Hh to which cells are exposed