The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras

Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.     

Distinct mechanisms determine the patterns of differential activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by receptors for growth factors or antigen

Although GTPases of the Ras family have been implicated in many aspects of the regulation of cells, little is known about the roles of individual family members. Here, we analyzed the mechanisms of activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by two types of external stimuli, growth factors and ligation of the antigen receptors of B or T lymphocytes (BCRs and TCRs). The growth factors interleukin-3, colony-stimulating factor 1, and epidermal growth factor all preferentially activated M-Ras and K-Ras 4B over H-Ras or N-Ras. Preferential activation of M-Ras and K-Ras 4B depended on the presence of their polybasic carboxy termini, which directed them into high-buoyant-density membrane domains where the activated receptors, adapters, and mSos were also present. In contrast, ligation of the BCR or TCR resulted in activation of H-Ras, N-Ras, and K-Ras 4B, but not M-Ras. This pattern of activation was not influenced by localization of the Ras proteins to membrane domains. Activation of H-Ras, N-Ras, and K-Ras 4B instead depended on the presence of phospholipase C-gamma and RasGRP. Thus, the molecular mechanisms leading to activation of Ras proteins vary with the stimulus and can be influenced by either colocalization with activated receptors or differential sensitivity to the exchange factors activated by a stimulus.     

Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells

Ras-membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras-lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein-tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.     

Regulating c-Ras function. cholesterol depletion affects caveolin association, GTP loading, and signaling.

Cholesterol-rich and caveolin-containing microdomains of the plasma membrane, termed "caveolae," have been implicated in signal transduction. However, the role of caveolae in regulating the Ras-MAP kinase cascade is incompletely understood. The mammalian Ras isoforms (H, N, and K) use different membrane anchors to attach to the plasma membrane and thereby may localize to functionally distinct microdomains, which might explain isoform-specific signaling. Here, we show that, in Cos epithelial cells, endogenous K-Ras colocalizes largely with caveolin, whereas N-Ras localizes to both caveolar and noncaveolar subdomains; H-Ras localization was below detection limits. We find that epidermal growth factor (EGF) activates N-Ras but fails to activate K-Ras in these cells. Extraction of cholesterol with methyl-beta-cyclodextrin disrupts complex formation between caveolin and K- and N-Ras and, strikingly, enables EGF to activate both K-Ras and N-Ras. While cholesterol depletion enhances GTP-loading on total c-Ras, activation of the downstream MEK-MAP kinase cascade by EGF and lysophosphatidic acid but not that by phorbol ester is inhibited. Thus, plasma membrane cholesterol is essential for negative regulation of c-Ras isoforms (complexed to caveolin), as well as for mitogenic signaling downstream of receptor-activated c-Ras.     

Nonconventional trafficking of Ras associated with Ras signal organization

Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.     

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.     

Downregulation of Ras C-terminal processing by JNK inhibition

After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked under JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.     

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

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.     

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics.     

H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment

H-, N-, and K-Ras are isoforms of Ras proteins, which undergo different lipid modifications at the C terminus. These post-translational events make possible the association of Ras proteins both with the inner plasma membrane and to the cytosolic surface of endoplasmic reticulum and Golgi complex, which is also required for the proper function of these proteins. To better characterize the intracellular distribution and sorting of Ras proteins, constructs were engineered to express the C-terminal domain of H- and K-Ras fused to variants of green fluorescent protein. Using confocal microscopy, we found in CHO-K1 cells that H-Ras, which is palmitoylated and farnesylated, localized at the recycling endosome in addition to the inner leaflet of the plasma membrane. In contrast, K-Ras, which is farnesylated and nonpalmitoylated, mainly localized at the plasma membrane. Moreover, we demonstrate that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins and that palmitoylation on this region of H-Ras might operate as a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells. Using selective photobleaching techniques, we demonstrate the dynamic nature of H-Ras trafficking to the recycling endosome from plasma membrane. We also provide evidence that Rab5 and Rab11 activities are required for proper delivery of H-Ras to the endocytic recycling compartment. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we found that a pool of GTP-bound H-Ras localized on membranes from Rab11-positive recycling endosome after serum stimulation. These results suggest that H-Ras present in membranes of the recycling endosome might be activating signal cascades essential for the dynamic and function of the organelle.     

Dual activation of phospholipase C-epsilon by Rho and Ras GTPases

Phospholipase C-epsilon (PLC-epsilon) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-epsilon to stimulate its phospholipase activity. Purified PLC-epsilon was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P(2)-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-epsilon prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-epsilon eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-epsilon markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383-394), but PLC-epsilon lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-epsilon by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-epsilon was quantified with PtdIns(4,5)P(2)-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-epsilon to activation by K-Ras. These results indicate that PLC-epsilon can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-epsilon.     

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.     

RIG1 inhibits the Ras/mitogen-activated protein kinase pathway by suppressing the activation of Ras

The retinoid-inducible gene 1 (RIG1) protein is a retinoid-inducible growth regulator. Previous studies have shown that the RIG1 protein inhibits the signaling pathways of Ras/mitogen-activated protein kinases. However, neither the mode of action nor the site of inhibition of RIG1 is known. This study investigated the effects of RIG1, and the mechanisms responsible for these effects, on the activation of Ras proteins in HtTA cervical cancer cells. RIG1 reduced the levels of activated Ras (Ras-GTP) and total Ras protein in cells transfected with mutated H-, N-, or K-Ras(G12V), or in cells transfected with the wild type H- or N-Ras followed by stimulation with epidermal growth factor. The half-life of Ras protein decreased from more than 36 h in control cells to 18 h in RIG1-transfected cells. RIG1 immunoprecipitated with the Ras protein in co-transfected cellular lysates. In contrast to the predominant plasma membrane localization in control cells, the H-Ras fusion protein EGFP-H-Ras was localized within a discrete cytoplasmic compartment where it co-localized with RIG1. RIG1 inhibited more than 93% of the Elk- and CHOP-mediated transactivation induced by H- or K-Ras(G12V). However, RIG1 did not inhibit the transactivation induced by MEK1 or MEK3, and failed to suppress the phosphorylation of extracellular signal-regulated kinases 1 and 2 induced by the constitutively activated B-Raf(V599E). The RIG1 with carboxyl terminal truncation (RIG1DeltaC) did not immunoprecipitate with Ras and had no effect on Ras activation or transactivation of the downstream signal pathways. These data indicate that RIG1 exerts its inhibitory effect at the level of Ras activation, which is independent of Ras subtype but dependent on the membrane localization of the RIG1 protein. This inhibition of Ras activation may be mediated through downregulation of Ras levels and alteration of Ras subcellular distribution.     

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.     

Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains

Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1.     

Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras

Galectin-3 (Gal-3) is a pleiotropic beta-galactoside-binding protein expressed at relatively high levels in human neoplasms. Its carbohydrate recognition domain (CRD) contains a hydrophobic pocket that can accommodate the farnesyl moiety of K-Ras. Binding of K-Ras to Gal-3 stabilizes K-Ras in its active (GTP-bound) state. Gal-3, which does not interact with N-Ras, was nevertheless shown to reduce N-Ras-GTP in BT-549 cells by an unknown mechanism that we explored here. First, comparative analysis of various cancer cell lines (glioblastomas, breast cancer cells and ovarian carcinomas) showed a positive correlation between low N-Ras-GTP/high K-Ras-GTP phenotype and Gal-3 expression levels. Next we found that epidermal growth factor-stimulated GTP loading of N-Ras, but not of K-Ras, is blocked in cells expressing high levels of Gal-3. Activation of Ras guanine nucleotide releasing proteins (RasGRPs) by phorbol 12-myristate 13-acetate (PMA) or downregulation of Gal-3 by Gal-3 shRNA increased the levels of N-Ras-GTP in Gal-3 expressing cells. We further show that the N-terminal domain of Gal-3 interacts with and inhibits RasGRP4-mediated GTP loading on N-Ras and H-Ras proteins. Growth of BT-549 cells stably expressing the Gal-3 N-terminal domain was strongly attenuated. Overall, these experiments demonstrate a new control mechanism of Ras activation in cancer cells whereby the Gal-3 N-terminal domain inhibits activation of N-Ras and H-Ras proteins.     

Targeting of K-Ras 4B by S-trans,trans-farnesyl thiosalicylic acid

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C(15) and C(20) S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras-membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.     

Long-term treatment with a beta-blocker timolol attenuates renal-damage in diabetic rats via enhancing kidney antioxidant-defense system

Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the translocation of RBD _Raf1 -GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems.     

Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity

Depending on the cellular context, Ras can activate characteristic effectors by mechanisms still poorly understood. Promotion by galectin-1 of Ras activation of Raf-1 but not of phosphoinositide 3-kinase (PI3-K) is one such mechanism. In this report, we describe a mechanism controlling selectivity of K-Ras4B (K-Ras), the most important Ras oncoprotein. We show that galectin-3 acts as a selective binding partner of activated K-Ras. Galectin-3 co-immunoprecipitated significantly better with K-Ras-GTP than with K-Ras-GDP, H-Ras, or N-Ras and colocalized with green fluorescent protein-K-Ras(G12V), not with green fluorescent protein-H-Ras(G12V), in the cell membrane. Co-transfectants of K-Ras/galectin-3, but not of H-Ras/galectin-3, exhibited enhanced and prolonged epidermal growth factor-stimulated increases in Ras-GTP, Raf-1 activity, and PI3-K activity. Extracellular signal-regulated kinase (ERK) activity, however, was attenuated in K-Ras/galectin-3 and in K-Ras(G12V)/galectin-3 co-transfectants. Galectin-3 antisense RNA inhibited the epidermal growth factor-stimulated increase in K-Ras-GTP but enhanced ERK activation and augmented K-Ras(G12V) transformation activity. Thus, unlike galectin-1, which prolongs Ras activation of ERK and inhibits PI3-K, K-Ras-GTP/galectin-3 interactions promote, in addition to PI3-K and Raf-1 activation, a third inhibitory signal that attenuates active ERK. These experiments established a novel and specific mechanism controlling the duration and selectivity of signals of active K-Ras, which is extremely important in many human tumors.     

Rasosomes originate from the Golgi to dispense Ras signals

Ras proteins undergo an incompletely understood trafficking process in the cell. Rasosomes are protein nanoparticles of 80–100 nm diameter that carry lipidated Ras isoforms (H-Ras and N-Ras) as well as their effectors through the cytoplasm and near the plasma membrane (PM). In this study, we identified the subcellular origin of rasosomes and how they spread Ras proteins through the cell. We found no dependency of rasosome formation on galectins, or on the GDP-/GTP-bound state of Ras. We found that significantly more rasosomes are associated with forms of Ras that are localized to the Golgi, namely N-Ras or the singly palmitoylated H-Ras mutant (C181S). To explore the possibility that rasosome originate from the Golgi, we used photoactivatable (PA)-GFP-H-Ras mutants and showed that rasosomes bud from the Golgi in a two-step mechanism. Newly released rasosomes first move in an energy-dependent directed fashion and then convert to randomly diffusing rasosomes. Dual fluorescence time-lapse imaging revealed the appearance of dually labeled rasosomes, indicating a dynamic exchange of cytoplasmic and PM-associated Ras with rasosome-associated Ras. Finally, higher levels of rasosomes correlate with higher levels of ERK phosphorylation, a key marker of Ras downstream signaling. We suggest that H-Ras and N-Ras proteins exchange with rasosomes that can function as carriers of palmitoylated Ras and its signals.