A Ceramide-binding C1 Domain Mediates Kinase Suppressor of Ras Membrane TranslocationYin X. · Zafrullah M. · Lee H. · Haimovitz-Friedman A. · Fuks Z. · Kolesnick R.
1Laboratory of Signal Transduction and2Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York
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.
Richard Kolesnick MD
Memorial Sloan-Kettering Cancer Center
1275 York Avenue, New York, NY 10021 (USA)
Tel. +1 646 888-2174, Fax: +1 646 422-0281
Accepted: May 23, 2009
Published online: August 03, 2009
Number of Print Pages : 12
Cellular Physiology and Biochemistry (International Journal of Experimental Cellular Physiology, Biochemistry andPharmacology)
Vol. 24, No. 3-4, Year 2009 (Cover Date: 2009)
Journal Editor: F. Lang, Tübingen
ISSN: 1015–8987 (Print), eISSN: 1421–9778 (Online)
For additional information: http://www.karger.com/journals/cpb