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

Abstract

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

Protein association changes in the Hedgehog signaling complex mediate differential signaling strength

“…To confirm this hypothesis, endogenous Smo in the dorsal compartment was depleted by RNA interference (RNAi), and the activity of SmoΔFu was tested in this new background. The double-strand RNA (dsRNA) sequence targeted against smo is located at the 5′ end of the smo transcript (Sanial et al, 2017), a sequence which is not present in the SmoΔFu transgene. The smo RNAi-dependent defects were shown to be fully rescued by a Smo-WT transgene (Sanial et al, 2017;Maier et al, 2014).…”

“…The double-strand RNA (dsRNA) sequence targeted against smo is located at the 5′ end of the smo transcript (Sanial et al, 2017), a sequence which is not present in the SmoΔFu transgene. The smo RNAi-dependent defects were shown to be fully rescued by a Smo-WT transgene (Sanial et al, 2017;Maier et al, 2014). To confirm this, we analyzed the activity of Smo Fu in a background depleted for gprk2 in which Hh signaling is less likely because Smo is not converted to a fully open conformation (Chen et al, 2010;Molnar et al, 2007).…”

“…Hh activation neutralizes the positively charged cluster by triggering the phosphorylation of an adjacent domain (Zhao et al, 2007). In Drosophila, this phosphorylation is sequential and triggered by several kinases that include protein kinase A (PKA; PKA-C1 -FlyBase), the serine-threonine kinase Fused (Fu) and G protein-coupled receptor kinase 2 (Gprk2), which promote the conversion of Smo to an open conformation (Zhang et al, 2004;Jia et al, 2004;Apionishev et al, 2005;Zhou et al, 2006;Jia et al, 2010;Chen et al, 2010;Fan et al, 2012;Jiang et al, 2014;Maier et al, 2014;Li et al, 2016;Sanial et al, 2017). This switch seems to be essential for the cell surface accumulation of Smo and the activation of signaling.…”

“…Fu consists of an N-terminal catalytic domain and a C-terminal regulatory domain (Fu-Reg). Fu-Reg interacts with several proteins of the HSC: Cos2 (Robbins et al, 1997;Sisson et al, 1997), Smo (Malpel et al, 2007Sanial et al, 2017) and PKA (Ranieri et al, 2014). Moreover, it has been proposed that Fu-Reg interacts with the Fu catalytic domain, blocking the kinase activity in the absence of Hh.…”

CBP/EP300-dependent acetylation and stabilization of HSF2 are compromised in the rare disorder, Rubinstein-Taybi syndrome

“…Cells were lysed in modified Laemmli buffer (5% SDS, 10% glycerol, 32.9 mM Tris-HCl pH 6.8) supplemented with 1 mM DTT (Sigma-Aldrich), and their extracts (15 µG) were run on 10% SDS-PAGE. Gels were then scanned on a Typhoon Trio imager (GE Healthcare; excitation 532 nm, emission 580 nm, PMT 700 V) for determination of signal intensity of the covalently-bound fluorescent products as described in (Sanial et al, 2017).…”