Impaired retrograde membrane traffic through endosomes in a mutant <scp>CHO</scp> cell defective in phosphatidylserine synthesis

Lee, Shoken; Uchida, Yoshishige; Emoto, Kazuo; Umeda, Masato; Kuge, Osamu; Taguchi, Tomohiko; Arai, Hiroyuki

doi:10.1111/j.1365-2443.2012.01622.x

Cited by 41 publications

(42 citation statements)

References 26 publications

(28 reference statements)

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“…1G). This reduction is comparable to the reduction in PtdSer levels observed in PSA-3 cells, which are defective in PtdSer synthesis when cultured in the absence of ethanolamine (22) (Fig. 1G).…”

Section: Resultssupporting

confidence: 64%

“…Total cellular lipids were isolated by Folch extraction (21). Lipid extracts, along with pure lipid standards, were spotted onto a high performance one-dimensional thin-layer chromatography (TLC) silica gel plate (catalog number 1057150001; EMD Millipore) and resolved by using methyl acetate-chloroform-1-propanolmethanol-1% aqueous KCl (25:25:25:10:9, vol/vol/vol/vol/vol) (22). After iodine labeling, lipid spots were cut out and quantified by phosphate analysis as described previously (23).…”

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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

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: Resultssupporting

confidence: 64%

mentioning

confidence: 99%

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

143

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“…For instance, in mammalian cell pulse-chase experiments, TGN38 and Shiga toxin have been shown to be retrograde cargos that traverse the recycling endosome en route to the TGN (20,21,25). Canonical recycling endosome regulators Rab11, FIP1/RCP, and mRme-1/EHD1 have been shown to be important regulators of recycling endosome-to-TGN transport of retrograde cargo (23,25,54).…”

Section: Discussionmentioning

confidence: 99%

“…For instance, CHO cell pulse-chase analysis of fusion proteins bearing the transmembrane and intracellular domains of retrograde recycling proteins, TGN38 and Furin, showed that TGN38 trafficked from the early endosome to recycling endosome to the Golgi, whereas Furin recycling involved transit from the early endosome to the late endosome to the Golgi (20,21). TGN38 and Shiga toxin have been shown to require distinct sets of SNARE proteins to complete transport to the Golgi, also indicating that different cargos recycle to the Golgi in different types of vesicles (22).…”

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confidence: 99%

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

Bai

Grant

2015

Proc. Natl. Acad. Sci. U.S.A.

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Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1-positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42-associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.retromer | endocytic recycling | endosome | toca | wave

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“…To further investigate the role of PS in mediating spatial cross talk between different Ras isoforms, we manipulated PM PS levels using a CHO mutant cell line, PSA-3, which lacks PS synthase 1 (PSS1) and is an ethanolamine auxotroph (38). Interestingly, the level of PS on the PMs of parental CHO cells was also dependent on the ethanolamine content of the culture media (Fig.…”

Section: Ras Nanoclusters Have Different Lipid Compositions But All mentioning

confidence: 99%

Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

Zhou

Luan

Rodkey

et al. 2014

Molecular and Cellular Biology

126

245

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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 ...

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Impaired retrograde membrane traffic through endosomes in a mutant CHO cell defective in phosphatidylserine synthesis

Cited by 41 publications

References 26 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

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

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