Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains

Roy, Sandrine; Luetterforst, Robert; Harding, Angus; Apolloni, Ann; Etheridge, Maria; Stang, Espen; Rolls, Barbara J.; Hancock, John F.; Parton, Robert G.

doi:10.1038/10067

Cited by 357 publications

(242 citation statements)

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“…Depletion of PM cholesterol by treatment with ␤-CD was previously shown to cause a failure of lateral segregation of H-Ras-GTP from H-Ras-GDP and to inhibit the signal output from the resulting aberrant H-Ras-GTP clusters (24,30,36,39). We observed exactly the same effect of fendiline on H-Ras lateral segregation, which readily accounts for the ability of fendiline to inhibit H-RasG12V signaling reported previously (16).…”

Section: Discussionsupporting

confidence: 86%

“…Significant differences (*, P Ͻ 0.05) were assessed by using Student's t tests. GTP) and H-RasG12V (H-Ras-GTP) nanoclustering are unaffected by cholesterol depletion (24,30), whereas cholesterol depletion selectively inhibits H-RasG12V signal output (24,30). This paradox was recently resolved by our finding that H-Ras-GTP and H-Ras-GDP lateral segregation fails in cholesterol-depleted cells, resulting in the assembly of H-Ras-GTP clusters with lipid raft components that impair MAPK activation (35,36,39,41).…”

Section: Resultsmentioning

confidence: 99%

“…(i) Sample preparation. MDCK cells stably expressing mGFP-K-Ras4BG12V (K-Ras4B with a Gly12-to-Val mutation) were treated with 10 M fendiline for 48 h. Crude cell membranes were prepared as described previously (24). The membrane pellet was resuspended in 150 mM ammonium bicarbonate.…”

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confidence: 99%

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Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

Cho

Hoeven

Zhou

et al. 2016

Molecular and Cellular Biology

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143

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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...

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Section: Discussionsupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

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Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

Cho

Hoeven

Zhou

et al. 2016

Molecular and Cellular Biology

Self Cite

143

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“…More importantly, TC10 but not Cdc42 contains two additional upstream cysteine residues that can undergo palmitoylation. The presence of these additional palmitoylation residues in TC10 is similar to that observed for H-Ras which are required for its localization to lipid raft microdomains [51,52]. In contrast, K-Ras contains a single cysteine that undergoes farnesylation but instead of any further cysteine modification contains a putative phospholipid binding polybasic stretch upstream of the CAAX motif that targets K-Ras to non-lipid raft domains in the plasma membrane.…”

Section: Tc10 and Lipid Raft Localizationmentioning

confidence: 59%

Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways

2002

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Insulin-stimulated glucose uptake in adipose tissue and striated muscle is critical for reducing post-prandial blood glucose concentrations and the dysregulation of this process is one hallmark of Type II (non-insulin-dependent) diabetes mellitus. It has been well established that the insulin-stimulated redistribution of the insulin responsive glucose transporter, GLUT-4, from intracellular storage sites to the plasma membrane depends on the production of phosphoinositide 3,4,5 trisphosphate by the Class IA Phosphatidylinositol 3' kinase. Recent discoveries however, have shown the presence of a second insulin signalling pathway leading to GLUT-4 translocation, a pathway dependent on insulin receptor signalling emanating from caveolae or lipid rafts at the plasma membrane. This pathway begins with the phosphorylation of the adaptor protein Cbl by the insulin receptor, and results in the activation of a small GTP binding protein, TC10, a member of the Rho family. TC10 is able to modulate actin structure in 3T3L1 adipocytes, and its overexpression inhibits insulin-stimulated GLUT-4 translocation, an inhibition completely dependent on localization of TC10 to the caveolae or lipid rafts. The spatial compartmentalization of insulin signalling from caveolae or lipid rafts provides a novel signalling pathway that functions in concert with general signalling mechanisms in the control of actin dynamics regulating insulin-dependent GLUT-4 translocation.

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“…While infections measure the collective effects of 2A pro damage during an infectious cycle, the data are entirely consistent with the per-enzyme rate kinetics observed in the RNA transfection assays. The nuclear import pathways studied here are also responsible for the import of a number of nuclear proteins (transportin 1, hnRNP A1, transportin 3, Srp20, importin ␣/␤ nucleolin, and PTB) that relocalize into the cytoplasm of poliovirus-and RV-infected cells and can stimulate viral replication and translation (8,10,(40)(41)(42)(43)(44)(45). Timed 2A pro disruption of these pathways could certainly affect overall viral replication levels, which contribute directly to RV virulence.…”

Section: Discussionmentioning

confidence: 95%

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

Watters

İnankur

Gardiner

et al. 2017

J Virol

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The RNA rhinoviruses (RV) encode 2A proteases (2A pro ) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2A pro sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2A pro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin ␣/␤, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2A pro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-B (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2A pro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2A pro ) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2A pro were measured comparatively in transformed cells programed with fluorescent reporter systems and by quantitative cell imaging, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were indeed attacked at different rates and with different affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses that directly or indirectly lead to distinct disease phenotypes.KEYWORDS 2A, live-cell imaging, nuclear export, nuclear localization, nuclear trafficking, protease, rhinovirus T he controlled trafficking of protein and RNA across the nuclear membrane is essential for many eukaryotic cellular processes. Nuclear pore complexes (NPC) are large proteinaceous channels embedded in the nuclear envelope that restrict and

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Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains

Cited by 357 publications

References 50 publications

Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases

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