Src family kinase activation in glycosphingolipid-rich membrane domains ofendothelial cells treated with oxidised low density lipoprotein

Extraction of ECV304 endothelial cells in 1% Triton X-100 at 4 degrees C re sulted in a detergent-insoluble pellet that contained 90% of the caveolin, 78% of the src family kinases and 99% of the annexin II. When detergent-tre ated cells were loaded beneath a 10-30% sucrose gradient the caveolin and a large proportion of the cellular cholesterol floated at a density of 1.09 g/cm(3), characteristic of caveolae and glycosphingolipid-rich membranes. W ith extended centrifugation the src family kinases, which were initially as sociated with this floating material, sedimented to the bottom of the gradi ent. Annexin II remained on the bottom of the gradient under both centrifug ation conditions. After 24-h incubation with oxidised low density lipoprote in (oxLDL) about 7.5% of the total sterol in the cells was replaced by 7-ke tocholesterol, the major oxysterol found in oxLDL. The majority of this 7-k etocholesterol was found in the light membrane fraction on sucrose gradient s. Under these conditions src kinase activity more than doubled in the Trit on-resistant fraction, without changes in the concentration of src kinase p rotein. Introducing oxysterols directly into the medium bathing ECV304 cell s for 1 h also modulated the activity of src family kinases in the detergen t-resistant membranes. An elevation in activity was observed for 7-ketochol esterol while 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol and cho lesterol epoxide all produced decreases in the background level of src kina se activity. We conclude that 7-ketocholesterol and possibly other componen ts of oxLDL can equilibrate into glycosphingolipid-rich membranes and incre ase the activity of src kinases, possibly by interaction with caveolin, (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.