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

Abstract

The 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.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused

“…Some conserved S and T are clustered in four groups (GI, II, III and IV) in the C-terminal. Binding to FU was reported to occur between aa 985 and 1036 (Malpel et al, 2007). SMO variants used in this study are shown below, where conserved S or T residues were substituted by alanine -HA (lanes 5 and 6) without (−) or with (+) GAP-gFU.…”

G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila

“…It is possible that the closed conformation state of Smo is also regulated by intermolecular interactions in addition to intramolecular interactions. For example, it has been shown that Fu can directly bind the Smo C terminus in the absence of Hh (Malpel et al 2007), and this interaction may help stabilize the closed conformation of Smo C-tail. Indeed, disrupting Smo/Fu interaction led to increased basal activity of Smo (Malpel et al 2007).…”

Fused–Costal2 protein complex regulates Hedgehog-induced Smo phosphorylation and cell-surface accumulation

“…For example, Cos2 may bind two distinct domains of Smo: one occupied in the absence of Hh and the other in the presence of Hh, with only one of the interactions being disrupted by Fu phosphorylation. Alternatively, Cos2 may bind directly with inactivated Smo, but associate with activated Smo indirectly through other protein(s) such as Fu, as it has been shown recently that Fu can bind directly to the C-terminal region of Smo in a yeast two-hybrid assay (Malpel et al 2007).…”