FIG. 7.

KSR1 couples Ras signals from lipid rafts to cPLA₂ activation. (A) Depletion of KSR1 prevents cPLA₂ but not RSK1 activation. 293T cells were transfected with (+) or without (-) a siRNA (10 nM) for KSR1 and stimulated with EGF (10 ng/ml, 5 min) after starvation. Endogenous cPLA₂ activation was determined by [³H]arachidonic acid release. Bottom panels show total and phosphorylated RSK1 (p-RSK1), and KSR1 levels were determined by immunoblotting. (B) Biphasic activation of cPLA₂ resulting from increasing KSR1 levels. Cells were transfected with increasing concentrations of FLAG-KSR1 and stimulated with EGF after starvation. Bottom panels show total and phosphorylated RSK1, and KSR1 (FLAG) levels were determined by immunoblotting. (A and B) Shown are the averages ± standard errors of the means of the results from three independent experiments relative to the levels in starved (C) cells. (C) ERK1/2-KSR1 association is enhanced by Ras signals from lipid rafts but not the disordered membrane. Endogenous ERK1/2, total and phosphorylated, present in native KSR1 immunoprecipitates was determined in cells transfected with RasV12 (0.5 μg) tethered to lipid rafts (LCK) or to the disordered membrane (CD8) or stimulated with EGF. (D) ERK1/2-KSR1 association is enhanced in wild-type but not in H/N-Ras^−/− MEFs. ERK1/2, total and phosphorylated, present in KSR1 immunoprecipitates were analyzed in MEFs, starved (C) or EGF stimulated. (E) The association of KSR1 and endogenous cPLA₂ is enhanced by EGF stimulation and by Ras signals from lipid rafts and is inhibited by UO126 (2 μM, 25 min prior to stimulation). Bottom panels, RSK1 does not associate with KSR1 in response to Ras signals coming from lipid rafts or to EGF stimulation. (F) The association of KSR1 and endogenous cPLA₂ in response to EGF is diminished in H/N-Ras^−/− MEFs compared to wild-type fibroblasts. TL, total lysate; IP, immunoprecipitations performed with a specific antibody; PI, immunoprecipitations performed with preimmune serum; α, anti.