Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling via Two Distinct Mechanisms

David J. Robbins,Mark M. Yore,Stacey K. Ogden,Manuel Ascano,David J. Casso,Thomas B. Kornberg

doi:10.1074/jbc.m510169200

Abstract

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci.

Highlights

In Drosophila, Hh-mediated target gene activation is thought to be a two-step process involving stabilization of Ci2 and an as yet uncharacterized activation step that converts cubitus interruptus (Ci) to a transcriptional activator [1,2,3,4,5,6]
DNA Constructs—Act-renilla was constructed by subcloning Renilla from the pRL-TK plasmid (Promega) into pAct 5.1A (Invitrogen) via ScaI and BsrbI restriction sites. pAct 5.1 ci was generated by subcloning ci from pUAS-ci [1] via KpnI restriction sites. pAct 5.1 3x HA-carboxyl-terminal Smo binding domain (CSBD) was generated by PCR amplifying base pairs 3001–3601 of Cos2 cDNA with primers that introduce BglII sites flanking the Cos2 coding sequence
We found that CSBD is a strong inhibitor of Hh-mediated activation of a ptc-luciferase reporter construct, capable of decreasing maximal Hh activation by nearly 80% (Fig. 1A)

Summary

Introduction

In Drosophila, Hh-mediated target gene activation is thought to be a two-step process involving stabilization of Ci2 and an as yet uncharacterized activation step that converts Ci to a transcriptional activator [1,2,3,4,5,6]. We and others [23,24,25,26] have reported that the cargo domain of Cos forms an association with Smo that is necessary for Hh-mediated Ci activation This likely occurs through Hh-mediated Smo stabilization that facilitates additional HSC to associate with Smo through Cos2mediated tethering [23]. This phenomenon is somewhat contrary to our observation that, following Hh activation, the bulk of Cos releases from cellular membranes [27], whereas Cos bound to Smo accumulates on the plasma membrane [15, 23, 26]. Our results suggest that Smo regulates two arms of the Hh pathway, repression and activation, independently of each other and that this regulation can be functionally separated through targeting the Cos2-cargo Smo interaction

Methods

Results

Conclusion