SUMMARY K-Ras is targeted to the plasma membrane by a C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain. We used quantitative spatial imaging and atomistic molecular dynamics simulations to examine molecular details of K-Ras plasma membrane binding. We found that the K-Ras anchor binds selected plasma membrane anionic lipids with defined head groups and lipid side chains. The precise amino acid sequence and prenyl group define a combinatorial code for lipid binding that extends beyond simple electrostatics; within this code lysine and arginine residues are non-equivalent and prenyl chain length modifies nascent polybasic domain lipid preferences. The code is realized by distinct dynamic tertiary structures of the anchor on the plasma membrane that govern amino acid side-chain-lipid interactions. An important consequence of this specificity is the ability of such anchors when aggregated to sort subsets of phospholipids into nanoclusters with defined lipid compositions that determine K-Ras signaling output.

f K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK ¡ PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. Ras proteins are small GTPases that regulate important cellular signaling cascades to control cell growth, proliferation, and differentiation (1). The three Ras isoforms, H-, N-, and K-Ras4B (here, K-Ras), are ubiquitously expressed in mammalian cells. Ras proteins must be localized to the inner leaflet of the plasma membrane (PM) by a C-terminal membrane anchor for biological activity. In the case of K-Ras, the anchor comprises a posttranslationally attached C-terminal cysteine farnesyl-methyl ester operating in concert with a polybasic motif of 6 lysine residues (2, 3). Electrostatic interactions between the K-Ras C-terminal polybasic domain and the negatively charged inner leaflet of the PM provide membrane affinity (2, 4-9). Maintenance of K-Ras on the PM also requires the chaperone protein PDE␦ (10). Cytosolic PDE␦ binds K-Ras released from the PM as a result of endocytosis and unloads K-Ras in the perinuclear region in response to Arl2/3 binding, whence K-Ras translocates to the recycling endosome (RE) for redelivery to the PM by vesicular transport (11). Ras proteins on the PM are spatially organized into nanodomains, called nanoclusters, that are required for high-fidelity signal transduction by the Ras/mitogen-activated protein kinase (MAPK) pathway (12-14). Ras GTP nanoclusters contain ϳ6 to 7 Ras proteins, are Ͻ20 nm in diameter, and are exclusive platforms for Raf recruitment and MEK/extracellular signal-regulated kinase (ERK) activation. Perturbation of the spatiotemporal dynamics of Ras nanoclustering disrupts cellular signaling (15, 16).We have used a high-content cell-based screen (HCS) to identify multiple chemical compounds that mislocalize K-Ras from the PM and abrogate K-Ras signal transmission (17,18). One group of compounds disrupts the cellular phosphatidylserine (PtdSer) distribution or PtdSer levels with a consequent reduction in the PtdSer content of the inner leaflet of the PM (18-21)....

Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.

KRAS must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and KRASG12V from the PM; resulting in abrogation of KRASG12V signaling and potent, selective growth inhibition of mutant KRAS transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic KRAS-expressing MiaPaCa-2 tumors, but had no effect on the growth of the wild-type KRAS-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a KRAS ortholog) signaling in as evidenced by suppression of the induced multi-vulva phenotype. Using RNAis against genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multi-vulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused KRASG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic KRAS-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for KRAS function, which may present therapeutic options for the treatment of KRAS-driven cancers.

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.