FIG. 10.

Sef couples Ras signals from the ER to cPLA₂ activation. (A) Depletion of KSR1 inhibits cPLA₂ activation by Ras signals from lipid rafts but not from the ER or disordered membrane. 293T cells were transfected with RasV12 (0.5 μg) tethered to lipid rafts, the ER, and the disordered membrane with (+) or without (−) a siRNA (10 nM) for KSR1. (B) Effects of depleting the levels of different scaffold proteins on cPLA₂ activation by Ras signals from the ER. Cells were transfected with ER-targeted RasV12 (M1) (+) plus siRNAs (10 nM) for the shown scaffold proteins. DGLY., dystroglycan; β ARR. 2, β-arrestin 2; PAXILL., paxillin. (C) cPLA₂ activation by Ras signals from the ER exhibits a biphasic response to increasing Sef concentrations. Cells were transfected with ER-targeted RasV12 (M1) (+), in addition to increasing concentrations of Myc-Sef as shown. (D) Sef does not intervene in cPLA₂ activation elicited from lipid rafts. Cells were transfected with RasV12 tethered to lipid rafts with (+) or without (-) a siRNA against Sef. (A to D) cPLA₂ activation was determined by [³H]arachidonic acid release after 18 h of starvation. Shown are the averages ± standard errors of the means of the results from three independent experiments relative to the levels in starved (C) cells. (E) Sef/cPLA₂ association is enhanced by Ras signals from the ER but not from lipid rafts. Endogenous cPLA₂, present in anti-Myc (α Myc) immunoprecipitates, was determined in cells transfected with Myc-Sef (1 μg) plus RasV12 tethered to lipid rafts (LCK) or to the ER (M1). The dependence on ERK was determined by incubation with UO126 (2 μM, 25 min prior to stimulation). TL, total lysate; IP, immunoprecipitations performed with a specific antibody; PI, immunoprecipitations performed with preimmune serum.