Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.
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.
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Xu
X
Xiao
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Jing-Jie
JJ
Tang
State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201203, China; Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 201204, China.
Chao
C
Peng
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China.
Yan
Y
Wang
State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Lin
L
Fu
Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China.
Zhi-Ping
ZP
Qiu
State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Yue
Y
Xiong
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China.
Lian-Fang
LF
Yang
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China.
Hai-Wei
HW
Cui
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China.
Xiao-Long
XL
He
Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China.
Lei
L
Yin
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Wei
W
Qi
State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201203, China; Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 201204, China.
Catherine C L
CC
Wong
State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Yun
Y
Zhao
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Bo-Liang
BL
Li
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China. Electronic address: wwqiu@chem.ecnu.edu.cn.
Wen-Wei
WW
Qiu
Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, China. Electronic address: blsong@whu.edu.cn.
Bao-Liang
BL
Song
D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D002923:Cilia / Q000378:metabolism; D003412:Cricetulus; D018507:Gene Expression Regulation, Developmental; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006330:Heart Defects, Congenital / Q000235:genetics / Q000378:metabolism; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D041681:NIH 3T3 Cells; D000072081:Patched-1 Receptor / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D015398:Signal Transduction; D000072086:Smoothened Receptor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D053823:Hedgehog Proteins; D000072081:Patched-1 Receptor; C000606240:Ptch1 protein, mouse; C491023:SMO protein, human; C465570:Shh protein, mouse; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D002784:Cholesterol
Hedgehog; Patched-1; Smoothened; cholesterol; cholesterylation; ciliary localization; click chemistry; cysteine-rich domain; embryonic lethal; primary cilia