RAS isoforms and mutations in cancer at a glance

RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP–GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts

Redox Regulation of Ras and Rho GTPases: Mechanism and Function

Significance: Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. Herein, we present evidence for the role of Ras and Rho GTPases in controlling these events and the unique underlying mechanisms. Evidence for redox regulation of Ras GTPases that contain a redox-sensitive cysteine (X) in the conserved NKXD motif is presented, and a growing consensus supports regulation by a thiyl radical-mediated oxidation mechanism. We also discuss the debate within the literature regarding whether 2e− oxidation mechanisms also regulate Ras GTPase activity. Recent Advances: We examine the increasing in vitro and cell-based data supporting oxidant-mediated activation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop (p-loop) motif (GXXXXG[S/T]C). While this motif is distinct from Ras, these data suggest a similar 1e− oxidation-mediated activation mechanism. Critical Issues: We also review the data showing that the unique p-loop placement of the redox-sensitive cysteine in Rho GTPases supports activation by 2e− cysteine oxidation. Finally, we examine the role that Ras and Rho GTPases play in controlling key oxidant-regulating enzymes in the cell, and we speculate on a feedback mechanism. Future Directions: Given that these GTPases and redox-regulating enzymes are involved in multiple physiological and pathological processes, we discuss future experiments that may clarify the interplay between them. Antioxid. Redox Signal. 18, 250–258

Glutathiolated Ras: Characterization and implications for Ras activation

Ras GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. Similar to the action of guanine-nucleotide exchange factors, the rate of GDP dissociation can be greatly enhanced by the reaction of Ras with small-molecule redox agents, such as nitrogen dioxide, which can promote Ras activation. Nitrogen dioxide is an auto-oxidation product of nitric oxide and can react with an accessible cysteine of Ras to cause oxidation of the bound guanine nucleotide to facilitate Ras guanine nucleotide dissociation. Glutathione has also been reported to modify Ras and alter its activity. To elucidate the mechanism by which glutathione alters Ras guanine nucleotide binding properties, we performed NMR and biochemical analyses of glutathiolated Ras. We determined that treatment of H-Ras, lacking the non-conserved hypervariable region, with oxidized glutathione results in glutathiolation specifically at cysteine 118. However, glutathiolation does not alter Ras structure or biochemical properties. Rather, changes in guanine nucleotide binding properties and Ras activity occur upon exposure of Ras to free radicals, presumably through the generation of a cysteine 118 thiyl radical. Interestingly, Ras glutathiolation protects Ras from further free radical-mediated activation events. Therefore, glutathiolation does not affect Ras activity unless Ras is modified by glutathione through a radical-mediated mechanism.

Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies

Researchers rely largely on antibodies to measure the abundance, activity, and localization of a protein, information that provides critical insight into both normal and pathological cellular functions. However, antibodies are not always reliable or universally valid for the methods in which they are used; in particular, the reliability of commercial antibodies against RAS is highly variable. Waters et al . rigorously assessed 22 commercially available RAS antibodies for their utility to detect the distinct RAS isoforms in various cell types and for their use in specific analytical methods. Their findings show how reliably one can interpret the data acquired from each reagent

Redox regulation of Rac1 by thiol oxidation

The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys18) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys18 by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1C18D. We also evaluated Rac1C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1WT but is resistant to thiol oxidation. In addition, Rac1C18D, but not Rac1C18S, shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1WT and the redox-insensitive Rac1C18S variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys18 is a novel posttranslational modification that upregulates Rac1 activity

Copper is required for oncogenic BRAF signalling and tumorigenesis

The BRAF kinase is mutated, typically V600E, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers1,2. BRAFV600E phosphorylates and activates the kinases MEK1 and MEK2, which in turn phosphorylate and activate the kinases ERK1 and ERK2, stimulating the MAPK pathway to promote cancer3. Targeting MEK1/2 is proving to be an important therapeutic strategy, as a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma4, which is increased when co-administered with a BRAFV600E inhibitor5. In this regard, we previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction6. We now show that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced BRAFV600E-driven signaling and tumorigenesis. Conversely, a MEK1-MEK5 chimera that phosphorylates ERK1/2 independent of Cu or an active ERK2 restored tumor growth to cells lacking Ctr1. Importantly, Cu chelators used in the treatment of Wilson disease7 reduced tumor growth of both BRAFV600E-transformed cells and cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers

Measurement of three-jet differential cross sections d sigma-3jet / d M-3jet in p anti-p collisions at sqrt(s)=1.96 TeV

We present the first measurement of the inclusive three-jet differential cross section as a function of the invariant mass of the three jets with the largest transverse momenta in an event in p anti-p collisions at sqrt(s) = 1.96 TeV. The measurement is made in different rapidity regions and for different jet transverse momentum requirements and is based on a data set corresponding to an integrated luminosity of 0.7 fb^{-1} collected with the D0 detector at the Fermilab Tevatron Collider. The results are used to test the three-jet matrix elements in perturbative QCD calculations at next-to-leading order in the strong coupling constant. The data allow discrimination between parametrizations of the parton distribution functions of the proton.Comment: 10 pages, 4 figures, 2 tables, submitted to Phys. Lett. B, corrected chi2 values for NNPD

Measurement of the production of a W boson in association with a charm quark in pp collisions at √s = 7 TeV with the ATLAS detector

The production of a W boson in association with a single charm quark is studied using 4.6 fb−1 of pp collision data at s√ = 7 TeV collected with the ATLAS detector at the Large Hadron Collider. In events in which a W boson decays to an electron or muon, the charm quark is tagged either by its semileptonic decay to a muon or by the presence of a charmed meson. The integrated and differential cross sections as a function of the pseudorapidity of the lepton from the W-boson decay are measured. Results are compared to the predictions of next-to-leading-order QCD calculations obtained from various parton distribution function parameterisations. The ratio of the strange-to-down sea-quark distributions is determined to be 0.96+0.26−0.30 at Q 2 = 1.9 GeV2, which supports the hypothesis of an SU(3)-symmetric composition of the light-quark sea. Additionally, the cross-section ratio σ(W + +c¯¯)/σ(W − + c) is compared to the predictions obtained using parton distribution function parameterisations with different assumptions about the s−s¯¯¯ quark asymmetry

State of the climate in 2013

In 2013, the vast majority of the monitored climate variables reported here maintained trends established in recent decades. ENSO was in a neutral state during the entire year, remaining mostly on the cool side of neutral with modest impacts on regional weather patterns around the world. This follows several years dominated by the effects of either La Niña or El Niño events. According to several independent analyses, 2013 was again among the 10 warmest years on record at the global scale, both at the Earths surface and through the troposphere. Some regions in the Southern Hemisphere had record or near-record high temperatures for the year. Australia observed its hottest year on record, while Argentina and New Zealand reported their second and third hottest years, respectively. In Antarctica, Amundsen-Scott South Pole Station reported its highest annual temperature since records began in 1957. At the opposite pole, the Arctic observed its seventh warmest year since records began in the early 20th century. At 20-m depth, record high temperatures were measured at some permafrost stations on the North Slope of Alaska and in the Brooks Range. In the Northern Hemisphere extratropics, anomalous meridional atmospheric circulation occurred throughout much of the year, leading to marked regional extremes of both temperature and precipitation. Cold temperature anomalies during winter across Eurasia were followed by warm spring temperature anomalies, which were linked to a new record low Eurasian snow cover extent in May. Minimum sea ice extent in the Arctic was the sixth lowest since satellite observations began in 1979. Including 2013, all seven lowest extents on record have occurred in the past seven years. Antarctica, on the other hand, had above-average sea ice extent throughout 2013, with 116 days of new daily high extent records, including a new daily maximum sea ice area of 19.57 million km2 reached on 1 October. ENSO-neutral conditions in the eastern central Pacific Ocean and a negative Pacific decadal oscillation pattern in the North Pacific had the largest impacts on the global sea surface temperature in 2013. The North Pacific reached a historic high temperature in 2013 and on balance the globally-averaged sea surface temperature was among the 10 highest on record. Overall, the salt content in nearsurface ocean waters increased while in intermediate waters it decreased. Global mean sea level continued to rise during 2013, on pace with a trend of 3.2 mm yr-1 over the past two decades. A portion of this trend (0.5 mm yr-1) has been attributed to natural variability associated with the Pacific decadal oscillation as well as to ongoing contributions from the melting of glaciers and ice sheets and ocean warming. Global tropical cyclone frequency during 2013 was slightly above average with a total of 94 storms, although the North Atlantic Basin had its quietest hurricane season since 1994. In the Western North Pacific Basin, Super Typhoon Haiyan, the deadliest tropical cyclone of 2013, had 1-minute sustained winds estimated to be 170 kt (87.5 m s-1) on 7 November, the highest wind speed ever assigned to a tropical cyclone. High storm surge was also associated with Haiyan as it made landfall over the central Philippines, an area where sea level is currently at historic highs, increasing by 200 mm since 1970. In the atmosphere, carbon dioxide, methane, and nitrous oxide all continued to increase in 2013. As in previous years, each of these major greenhouse gases once again reached historic high concentrations. In the Arctic, carbon dioxide and methane increased at the same rate as the global increase. These increases are likely due to export from lower latitudes rather than a consequence of increases in Arctic sources, such as thawing permafrost. At Mauna Loa, Hawaii, for the first time since measurements began in 1958, the daily average mixing ratio of carbon dioxide exceeded 400 ppm on 9 May. The state of these variables, along with dozens of others, and the 2013 climate conditions of regions around the world are discussed in further detail in this 24th edition of the State of the Climate series. © 2014, American Meteorological Society. All rights reserved