A ceramide-binding C1 domain mediates kinase suppressor of Ras membrane translocation Journal Article
| Authors: | Yin, X.; Zafrullah, M.; Lee, H.; Haimovitz-Friedman, A.; Fuks, Z.; Kolesnick, R. |
| Article 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. Copyright © 2009 S. Karger AG, Basel. |
| Keywords: | signal transduction; epidermal growth factor; controlled study; unclassified drug; nonhuman; protein domain; protein motif; animal cell; animals; protein kinases; fluorescent dyes; protein binding; enzyme activation; cercopithecus aethiops; cos cells; enzyme linked immunosorbent assay; recombinant fusion proteins; protein purification; gene disruption; escherichia coli; cell membrane; protein transport; glutathione transferase; protein structure, tertiary; binding sites; ganglioside gm1; ras proteins; genes, ras; protein structure; indoles; diacylglycerol; phosphatidylcholine; enzyme mechanism; fluorescent antibody technique, indirect; regulator protein; ceramide; ceramides; cell membrane transport; glycosphingolipid; mammal cell; c1 domain; ksr1; ceramide derivative; cysteic acid; kinase suppressor of ras 1; sphingomyelin; intracellular space; second messenger; transport kinetics; proto-oncogene proteins c-raf |
| Journal Title: | Cellular Physiology and Biochemistry |
| Volume: | 24 |
| Issue: | 3-4 |
| ISSN: | 1015-8987 |
| Publisher: | Cell Physiol Biochem Press |
| Date Published: | 2009-01-01 |
| Start Page: | 219 |
| End Page: | 230 |
| Language: | English |
| DOI: | 10.1159/000233248 |
| PUBMED: | 19710537 |
| PROVIDER: | scopus |
| PMCID: | PMC2978518 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 5" - "Export Date: 30 November 2010" - "CODEN: CEPBE" - "Source: Scopus" |
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