Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling

Mol Cell. 2017 Apr 6;66(1):154-162.e10. doi: 10.1016/j.molcel.2017.02.015. Epub 2017 Mar 23.

Abstract

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Keywords: Hedgehog; Patched-1; Smoothened; cholesterol; cholesterylation; ciliary localization; click chemistry; cysteine-rich domain; embryonic lethal; primary cilia.

MeSH terms

  • Animals
  • CHO Cells
  • Cholesterol / metabolism*
  • Cilia / metabolism
  • Cricetulus
  • Gene Expression Regulation, Developmental
  • Genetic Predisposition to Disease
  • HEK293 Cells
  • Heart Defects, Congenital / genetics
  • Heart Defects, Congenital / metabolism
  • Hedgehog Proteins / genetics
  • Hedgehog Proteins / metabolism*
  • Humans
  • Mice
  • Mice, Transgenic
  • Mutation
  • NIH 3T3 Cells
  • Patched-1 Receptor / genetics
  • Patched-1 Receptor / metabolism
  • Phenotype
  • Protein Processing, Post-Translational
  • RNA Interference
  • Signal Transduction*
  • Smoothened Receptor / genetics
  • Smoothened Receptor / metabolism*
  • Transfection

Substances

  • Hedgehog Proteins
  • Patched-1 Receptor
  • Ptch1 protein, mouse
  • SMO protein, human
  • Shh protein, mouse
  • Smo protein, mouse
  • Smoothened Receptor
  • Cholesterol