Inhibition of acid sphingomyelinase depletes cellular phosphatidylserine and mislocalizes K-Ras from the plasma membrane
K-Ras must localize to the plasma membrane for biological activity, thus preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both K-Ras isoforms, K-Ras4A and K-Ras4B, from the plasma membrane to endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM-inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely supplementation with exogenous cholesterol restores K-Ras4A, but not K-Ras4B, nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly delivery of recombinant ASM, or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick Type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization.