title: A ceramide-binding C1 domain mediates kinase suppressor of ras membrane translocation.
abstract: Genetic and biochemical data support Kinase Suppressor of Ras 1 (KSR1) as a positive regulator of the Ras-Raf-MAPK pathway, functioning as a kinase and/or scaffold to regulate c-Raf-1 activation. Membrane translocation mediated by the KSR1 CA3 domain, which is homologous to the atypical PKC C1 lipid-binding domain, is a critical step of KSR1-mediated c-Raf-1 activation. In this study, we used an ELISA to characterize the KSR1 CA3 domain as a lipid-binding moiety. Purified GST-KSR1-CA3 protein effectively binds ceramide but not other lipids including 1,2-diacylglyceol, dihydroceramide, ganglioside GM1, sphingomyelin and phosphatidylcholine. Upon epidermal growth factor stimulation of COS-7 cells, KSR1 translocates into and is activated within glycosphingolipid-enriched plasma membrane platforms. Pharmacologic inhibition of ceramide generation attenuates KSR1 translocation and KSR1 kinase activation in COS-7 cells. Disruption of two cysteines, which are indispensable for maintaining ternary structure of all C1 domains and their lipid binding capability, mitigates ceramide-binding capacity of purified GST-KSR1-CA3 protein, and inhibits full length KSR1 membrane translocation and kinase activation. These studies provide evidence for a mechanism by which the second messenger ceramide can target proteins to subcellular compartments in the process of transmembrane signal transduction.
authors.affiliation: Laboratory of Signal Transduction, Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
authors.forename: Xianglei
authors.initials: X
authors.lastname: Yin
authors.forename: Mohammad
authors.initials: M
authors.lastname: Zafrullah
authors.forename: Hyunmi
authors.initials: H
authors.lastname: Lee
authors.forename: Adriana
authors.initials: A
authors.lastname: Haimovitz-Friedman
authors.forename: Zvi
authors.initials: Z
authors.lastname: Fuks
authors.forename: Richard
authors.initials: R
authors.lastname: Kolesnick
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D011905:Genes, ras; D005982:Glutathione Transferase / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002518:Ceramides; D005456:Fluorescent Dyes; D007211:Indoles; D011993:Recombinant Fusion Proteins; C007293:DAPI; D005982:Glutathione Transferase; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins