title: Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.
abstract: K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization.
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Medical School, Houston, Texas, USA.
authors.forename: Kwang-Jin
authors.initials: KJ
authors.lastname: Cho
authors.affiliation: Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry, Houston, Texas, USA.
authors.forename: Dharini
authors.initials: D
authors.lastname: van der Hoeven
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Medical School, Houston, Texas, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada Departments of Surgery and Biochemistry, University of Toronto, Toronto, Ontario, Canada Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Ehime, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Maekawa
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Medical School, Houston, Texas, USA.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Medical School, Houston, Texas, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, Ontario, Canada Departments of Surgery and Biochemistry, University of Toronto, Toronto, Ontario, Canada fairng@smh.ca john.f.hancock@uth.tmc.edu.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Fairn
authors.affiliation: Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Medical School, Houston, Texas, USA fairng@smh.ca john.f.hancock@uth.tmc.edu.
authors.forename: John F
authors.initials: JF
authors.lastname: Hancock
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005275:Fendiline / Q000494:pharmacology*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase; D018631:ras Proteins; D005275:Fendiline