Phosphatidic acid binding to Patched contributes to the inhibition of Smoothened and Hedgehog signaling in Drosophila wing development

Jie Zhang, Yajuan Liu, Chi Wang, Craig W. Vander Kooi, Jianhang Jia

Research output: Contribution to journalArticlepeer-review

Abstract

Hedgehog (Hh) signaling controls growth and patterning during embryonic development and homeostasis in adult tissues. Hh binding to the receptor Patched (Ptc) elicits intracellular signaling by relieving Ptc-mediated inhibition of the transmembrane protein Smoothened (Smo). We uncovered a role for the lipid phosphatidic acid (PA) in the regulation of the Hh pathway in Drosophila melanogaster. Deleting the Ptc C-terminal tail or mutating the predicted PA-binding sites within it prevented Ptc from inhibiting Smo in wing discs and in cultured cells. The C-terminal tail of Ptc directly interacted with PA in vitro, an association that was reduced by Hh, and increased the amount of PA at the plasma membrane in cultured cells. Smo also interacted with PA in vitro through a binding pocket located in the transmembrane region, and mutating residues in this pocket reduced Smo activity in vivo and in cells. By genetically manipulating PA amounts in vivo or treating cultured cells with PA, we demonstrated that PA promoted Smo activation. Our findings suggest that Ptc may sequester PA in the absence of Hh and release it in the presence of Hh, thereby increasing the amount of PA that is locally available to promote Smo activation.

Original languageEnglish
Article numbereadd6834
JournalScience Signaling
Volume16
Issue number807
DOIs
StatePublished - Oct 17 2023

Bibliographical note

Publisher Copyright:
Copyright © 2023 The Authors, some rights reserved.

Funding

Acknowledgments:W earegratefultoG.DufortheGFP-PASSconstruct;M.Lehmannforthe dLipinanddLipin△PAPflies;J.JiangfortheMARCMfliesandMARCMcombinedwithC765-Gal4 flies;BloomingtonDrosophilaStockCenter(BSC)andViennaDrosophilaResourceCenter (VDRC)forflystocks;andtheDevelopmentalStudiesHybridomaBank(DSHB)fortheanti-Ci andanti–β-tubulinantibodies.W ealsothankF .YanforhelpwithgeneratingtheSev-PtcC construct during manuscript revision. Funding: This study was supported by the National InstitutesofHealthgrants(MIRAR35GM131807)toJ.J.Thisstudywasalsosupportedbythe SharedResourceFacilitiesoftheUniversityofKentuckyMarkeyCancerCenter(P30CA177558) andtheImagingCoreandPilotfundsfromtheCOBRE(P20GM121327).Authorcontributions: Conceivedanddesignedtheexperiments:J.J.,J.Z.,andY .L. Performedtheexperiments:J.Z.,Y .L., andJ.J.Analyzedthedata:J.Z.,C.W .V .K., C.W ., andJ.J.Wr ote thepaper:J.J.Competing interests:Theauthorsdeclarethattheyhav enocompetinginterests.Dataandmaterials availability:Alldataneededtoevaluatetheconclusionsinthepaperarepresentinthepaper ortheSupplementaryMaterials. We are grateful to G. Du for the GFP-PASS construct; M. Lehmann for the dLipin and dLipin∆PAP flies; J. Jiang for the MARCM flies and MARCM combined with C765-Gal4 flies; Bloomington Drosophila Stock Center (BSC) and Vienna Drosophila Resource Center (VDRC) for fly stocks; and the Developmental Studies Hybridoma Bank (DSHB) for the anti-Ci and anti–β-tubulin antibodies. We also thank F. Yan for help with generating the Sev-PtcC construct during manuscript revision. This study was supported by the National Institutes of Health grants (MIRA R35GM131807) to J.J. This study was also supported by the Shared Resource Facilities of the University of Kentucky Markey Cancer Center (P30CA177558) and the Imaging Core and Pilot funds from the COBRE (P20GM121327).

FundersFunder number
CEPR COBRE
National InstitutesofHealthgrantsP20GM121327, MIRAR35GM131807
National Institutes of Health (NIH)MIRA R35GM131807
National Institutes of Health (NIH)
University of Kentucky Markey Cancer CenterP30CA177558
University of Kentucky Markey Cancer Center

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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