Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

e K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks KRas signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the 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. Ras proteins are small guanine nucleotide binding proteins that oscillate between active GTP-bound and inactive GDP-bound states. Activated Ras proteins transmit signals for cell proliferation and cell survival. Importantly, ϳ15% of all human tumors express mutant Ras proteins that are locked in the GTP-bound state (1). Of the three ubiquitously expressed Ras isoforms, H-, N-, and K-Ras, oncogenic mutant K-Ras is the most prevalent, being expressed in ϳ95% of pancreatic, ϳ45% of colorectal, and ϳ35% of lung cancers (1). Despite its importance, there are currently no clinically approved drugs that directly target oncogenic K-Ras. To date, Ras drug discovery efforts have focused largely on inhibitors of Ras downstream effectors, including B-Raf, C-Raf, phosphatidylinositol 3-kinase (PI3K), MEK, and extracellular signal-regulated kinase (ERK) (2). For example, B-Raf-specific inhibitors produce excellent albeit often short-lived responses in patients with B-Raf mutant melanoma (3), in part because of a perturbation of complex negative-feedback control loops (2). B-Raf inhibitors also paradoxically activate the mitogen-activated protein kinase (MAPK) cascade in melanoma cells expressing oncogenic mutant N-or K-Ras (4-6). Other highly promising approaches include compounds that covalently modify K-Ras proteins with a G12C mutation to abrogate effector interactions (7,8) and allosteric modulators that directly bind Ras to inhibit guanine nucleotide exchange factor (GEF)-mediated nucleotide exchange (9-11). Chronic i...

Background: Ras proteins must be plasma membrane-localized for biological activity. Results: A high content screen identified staurosporines as inhibitors of Ras plasma membrane localization and K-Ras signal transmission by disrupting endosomal recycling of phosphatidylserine. Conclusion: Staurosporines are novel inhibitors of phosphatidylserine trafficking. Significance: Ras trafficking pathways and Ras spatiotemporal organization on the plasma membrane are valid targets for anti-Ras drug development.

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.