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.

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here, we describe a functionally critical region of RAS located outside the effector lobe that can be targeted for inhibition. We developed a synthetic binding protein (monobody), termed NS1, that bound with high affinity to both GTP- and GDP-bound states of H- and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS disrupting RAS dimerization/nanoclustering, which in turn blocked CRAF:BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells (CPCs) distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart [1–3]. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract (OFT) and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary OFT but not a right ventricle [4], the existence and function of SHF-like cells in these species has remained a topic of speculation [4–10]. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss of function studies in zebrafish, a lower vertebrate with a single ventricle, that latent-TGFβ binding protein 3 (ltbp3) transcripts mark a field of CPCs with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3+ cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the OFT and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGFβ ligands [11], zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of CPCs in both fields [4]. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3+ cells due to compromised progenitor proliferation. Additionally, small-molecule inhibition of TGFβ signaling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGFβ type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGFβ signaling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Ras proteins assemble into transient nanoclusters on the plasma membrane. Nanoclusters are the sites of Ras effector recruitment and activation and are therefore essential for signal transmission. The dynamics of nanocluster formation and disassembly result in interesting emergent properties including high-fidelity signal transmission. More recently the lipid structure of Ras nanoclusters has been reported and shown to contribute to isoform-specific Ras signaling. In addition specific lipids play critical roles in mediating the formation, stability and dynamics of Ras nanoclusters. In consequence the spatiotemporal organization of these lipids has emerged as important and novel regulators of Ras function. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

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.

K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15-20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1-3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.

b Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing. H-, N-, and K-Ras are small GTPases that operate as molecular switches to regulate cell growth, proliferation, and differentiation (1, 2). H-, N-, and K-Ras comprise nearly identical G domains (amino acids 1 to 165), which bind guanine nucleotides and interact with effectors and exchange factors, but they contain highly divergent C-terminal hypervariable regions (HVRs) (3, 4). The HVR undergoes posttranslational processing to attach a membrane anchor, which consists of a C-terminal S-farnesyl cysteine carboxylmethyl ester (common to all Ras proteins) and mono-palmitoylation of N-Ras, di-palmitoylation of H-Ras, and the presence of a polylysine domain in K-Ras (5-8). Ras proteins are distributed heterogeneously over the plasma membrane (PM) in a combination of immobile nanoclusters and freely diffusing monomers (9). A nanocluster comprises ϳ7 Ras proteins, has a radius of ϳ9 nm, and has an estimated lifetime of 0.5 to 1 s (10, 11). Nanocluster formation is essential for high-fidelity signal transmission (11-16). As a direct consequence of the different lipid anchors and different residues in the flanking HVR and G domain, which directly participate in membrane binding, N-, H-, and K-Ras assemble into spatially nonoverlapping nanoclusters, with further lateral segregation into nonoverlapping GDP and GTP nanoclusters (11,(16)(17)(18)(19)(20)(21)(22)(23).Ras isoforms exhibit different effector activation profiles (1). To account for isoform-specific signal output, Ras ...

Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor’s capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.