NMR <Superscript>1</Superscript>H,<Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and <Superscript>13</Superscript>C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here ¹H^{N, 15}N, and ¹³C resonance assignments for the 19.3 kDa (aa 1–169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS^G12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone ¹H–¹⁵N correlations have been assigned for all non-proline residues, except for the first methionine residue.     

Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

Background

Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells.

Methodology/Principal Findings

Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras^−/−,N-ras^−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras^−/−) embrionary fibroblasts by mating of K-ras^+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras^−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras^−/− fibroblasts.

Conclusions/Significance

We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations.     

Specificity in Ras and Rap Signaling

Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.Ras-like small G-proteins are ubiquitously expressed, conserved molecular switches that couple extracellular signals to various cellular responses. Different signals can activate GEFs² that induce the small G-protein to switch from the inactive, GDP-bound state to the active, GTP-bound state. This induces a conformational change that allows downstream effector proteins to bind specifically to and be activated by the GTP-bound protein to mediate diverse biological responses. Small G-proteins are returned to the GDP-bound state by hydrolyzing GTP with the help of GAPs. Ras (Ha-Ras, Ki-Ras, and N-Ras) and Rap proteins (Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C) have similar effector-binding regions that interact predominantly with RA domains or the structurally similar RBDs present in a variety of different proteins. Both protein families operate in different signaling networks. For instance, Ras is central in a network controlling cell proliferation and cell survival, whereas Rap1 predominantly controls cell adhesion, cell junction formation, cell secretion, and cell polarity. These different functions are reflected in a largely different set of GEFs and GAPs. Also the downstream effector proteins operate in a selective manner in either one of the networks.     

High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.     

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B,Exposing the Effector Binding Site

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B^WT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B^WT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition

Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H₂O, exchange reactions can lead to contamination of ²H sites by ¹H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing ¹H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U–²H, ¹⁵N]-chlorella ubiquitin without and with added inhibitors, and [U–¹⁵N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U–¹³C, ¹⁵N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at C^α sites, with the exception of Gly, and at C^β sites of Ala. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Asn–H^β, Asp–H^β, Gln–H^γ, Glu–H^γ, and Lys–H^ε. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of interest in studies of large proteins, protein complexes, and membrane proteins.     

Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch

Summary 1. Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane (PM) microdomains and various intracellular compartments. Such interaction is dictated by individual elements in the carboxy-terminal domain of the Ras proteins, including a farnesyl isoprenoid group, sequences in the hypervariable region (hvr)-linker, and palmitoyl groups in H/N-Ras isoforms.2. The farnesyl group acts as a specific recognition unit that interacts with prenyl-binding pockets in galectin-1 (Gal-1), galectin-3 (Gal-3), and cGMP phosphodiesterase δ. This interaction appears to contribute to the prolongation of Ras signals in the PM, the determination of Ras effector usage, and perhaps also the transport of cytoplasmic Ras. Gal-1 promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3-K) and Ral guanine nucleotide exchange factor (RalGEF), while galectin-3 promotes K-Ras signaling to both Raf and PI3-K.3. The hvr-linker and the palmitates of H-Ras and N-Ras determine the micro- and macro-localizations of these proteins in the PM and in the Golgi, as well as in ‘rasosomes’, randomly moving nanoparticles that carry palmitoylated Ras proteins and their signal through the cytoplasm.4. The dynamic compartmentalization of Ras proteins contributes to the spatial organization of Ras signaling, promotes redistribution of Ras, and provides an additional level of selectivity to the signal output of this regulatory GTPase.     

CPMG relaxation dispersion NMR experiments measuring glycine 1Hα and 13Cα chemical shifts in the ‘invisible’ excited states of proteins

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (>0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of ¹H^α and ¹³C^α chemical shifts of glycine residues in the excited state that reflects the fact that in this case the ¹H^α, ¹³C^α spins form a three-spin system that is more complex than the AX ¹H^α–¹³C^α spin systems in the other amino acids. Here pulse sequences for recording ¹H^α and ¹³C^α CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which ¹H^α, ¹³C^α chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.     

Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state

Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich nuclear targeting signals

Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. In addition to the known role in tumorigensis, the activated form of Ras exhibits growth-inhibitory effects by unknown mechanisms. Several Ras effector proteins identified as mediators of apoptosis and cell-cycle arrest also exhibit properties normally associated with tumor suppressor proteins. Here, we show that Ras effector RASSF5/NORE-1 binds strongly to K-Ras but weakly to both N-Ras and H-Ras. RASSF5 was found to localize both in the nucleus and the nucleolus in contrast to other Ras effector proteins, RASSF1C and RASSF2, which are localized in the nucleus and excluded from nucleolus. A 50 amino acid residue transferable arginine-rich nucleolar localization signal (NoLS) identified in RASSF5 is capable of interacting with importin-beta and transporting the cargo into the nucleolus. Surprisingly, similar arginine-rich signals identified in RASSF1C and RASSF2 interact with importin-alpha and transport the heterologous cytoplasmic proteins to the nucleus. Interestingly, mutation of arginine residues within these nuclear targeting signals prevented interaction of Ras effector proteins with respective transport receptors and abolished their nuclear translocation. These results provide evidence for the first time that arginine-rich signals are able to recognize different nuclear import receptors and transport the RASSF proteins into distinct sub-cellular compartments. In addition, our data suggest that the nuclear localization of RASSF5 is critical for its cell growth control activity. Together, these data suggest that the transport of Ras effector superfamily proteins into the nucleus/nucleolus may play a vital role in modulating Ras-mediated cell proliferation during tumorigenesis.     

Accurate measurements of the effects of deuteration at backbone amide positions on the chemical shifts of 15N, 13Cα, 13Cβ, 13CO and 1Hα nuclei in proteins

An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins (¹⁵N, ¹³C_α, ¹³CO, ¹H_α) and ¹³C_β nuclei arising from ¹H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond ¹³C_α isotope shifts, ²ΔC_α(N_iD) and ³ΔC_α(N_i+1D), and three-bond ¹³C_β isotope shifts, ³ΔC_β(N_iD), are useful reporters of the local geometry of the protein backbone.     

Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein

Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone ¹H^N, ¹⁵N, ¹³C′, and ¹³C^α, as well as side chain ¹³C^β, methyl (Ile-δ1, Leu, Val), amide (Asn, Gln), and indole N–H (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.     

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-¹³C₆,²H₇]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with ¹³C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-¹³C₆,²H₇]glucose.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments of light organ-associated fatty acid-binding protein of Taiwanese fireflies

Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C₁₄–C₁₈. However, the structural details are unknown, and the structure–function relationship has remained to be further investigated. In this study, we finished the ¹H, ¹⁵N and ¹³C chemical shift assignments of ¹⁵N/¹³C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, C_α, H_α, C and side chain C_β assignments. These results can provide the basis for further structural exploration of lcFABP.     

PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes

The cyclopentenone prostaglandin A₁ (PGA₁) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA₁ triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA₁ treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA₁, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA₁ evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.The mammalian genome contains three ras genes that encode the 21-kDa proteins H-Ras, N-Ras, and K-Ras with their two isoforms, K-Ras4A and K-Ras4B, which are generated from two alternative fourth exons. Ras proteins are small GTPases that act as molecular switches connecting a wide spectrum of extracellular signals from cell-surface receptors to intracellular pathways to control cell proliferation, differentiation, senescence, and death.¹ Ras proteins are activated by guanine nucleotide-exchange factors, which promote the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP), resulting in a conformational change of the tertiary structure of Ras and exposing its effector loop to interacting partners. The intrinsic GTPase activity of Ras proteins stimulated by GTPase-activating proteins restores the GDP-bound state and terminates Ras signaling. Active GTP-bound Ras interacts with effector proteins that modulate different signaling pathways to generate specific biological outcomes. As Ras proteins are ubiquitously expressed (except K-Ras4A)² and share a high degree of sequence homology and a large number of molecular activators, it was long assumed that their role was redundant. However, functional redundancy is not complete, as demonstrated by embryonic lethality in K-Ras knockouts^{3, 4} and the demonstration that the three Ras proteins have specific roles according to Ras isoform-dependent subcellular compartmentalization.⁵ This observation implies that biochemical and biophysical aspects of specific subcellular sites determine both the Ras isoform and the set of effectors that could be recruited, thus generating different molecular and biological outputs.⁶ Cyclopentenone prostaglandins (CyPGs) are eicosanoids with a varied spectrum of biological activity, including anti-inflammatory and antitumor effects, induction of oxidative stress, modulation of heat-shock response (HSP), and anti-viral activity.^{7, 8} They are thought to originate from the free radical-induced peroxidation of arachidonic acid (isoprostane pathway)⁹ and the dehydration of prostaglandins.¹⁰ CyPG contain an α,β-unsaturated carbonyl group in the cyclopentane ring that favors the formation of Michael adducts with sulfhydryl groups of proteins.¹¹ This mechanism is responsible for many of the biological properties of these compounds. However, the variety of cellular responses to CyPG appears to be cell type-specific and concentration-dependent; indeed, cytoprotective and cytotoxic responses have been associated with low and high concentrations of CyPG, respectively.^{12, 13} In this regard, we reported that H-Ras is a target for the addition of a typical CyPG, the 15-deoxy-12,14-prostaglandin J₂ (15d-PGJ₂), and that this effect is associated with specific activation of H-Ras-dependent pathways and increased proliferation and inhibition of apoptosis in NIH3T3 fibroblasts¹⁴ and MCA3D keratinocytes.¹⁵ Nevertheless, members of the prostaglandin A and J series act as potent inhibitors of some human tumors both in vitro and in vivo, by inducing cell cycle arrest and apoptosis.^{7, 10} In endothelial cells, activation of peroxisome proliferator-activated receptor (PPAR) receptors by 15d-PGJ₂ induces nuclear localization of receptors and caspase-mediated apoptosis,¹⁶ and both 15d-PGJ₂ and cyclopentenone prostaglandin A1 (PGA₁) induce apoptosis in AGS cells by a PPAR-independent mechanism.¹⁷ Although the role of CyPG as inducers of apoptosis is well documented, the relationship between Ras activation by CyPG and triggering of apoptosis, or proliferation, is not fully understood. We have explored the molecular mechanisms underlying PGA₁-induced apoptosis. We found that PGA₁ induces apoptosis in mouse fibroblasts by specific binding to and activation of H- and N-Ras at endomembranes, and that this process requires activation of extracellular signal-regulated kinases (ERKs). A detailed analysis revealed that the mechanism of cell death induced by PGA₁ requires ERK-mediated activation of calpain, an endoplasmic reticulum (ER) protease, and leads to a caspase-dependent apoptosis. Finally, we present evidence that the mechanism of apoptosis is shared by human cancer cells.     

Regulation of Na(+) reabsorption by the aldosterone-induced small G protein K-Ras2A

Xenopus laevis A6 cells were used as model epithelia to test the hypothesis that K-Ras2A is an aldosterone-induced protein necessary for steroid-regulated Na(+) transport. The possibility that increased K-Ras2A alone is sufficient to mimic aldosterone action on Na(+) transport also was tested. Aldosterone treatment increased K-Ras2A protein expression 2.8-fold within 4 h. Active Ras is membrane associated. After aldosterone treatment, 75% of K-Ras was localized to the plasma membrane compared with 25% in the absence of steroid. Aldosterone also increased the amount of active (phosphorylated) mitogen-activated protein kinase kinase likely through K-Ras2A signaling. Steroid-induced K-Ras2A protein levels and Na(+) transport were decreased with antisense K-ras2A oligonucleotides, showing that K-Ras2A is necessary for the natriferic actions of aldosterone. Aldosterone-induced Na(+) channel activity, was decreased from 0.40 to 0.09 by pretreatment with antisense ras oligonucleotide, implicating the luminal Na(+) channel as one final effector of Ras signaling. Overexpression of K-Ras2A increased Na(+) transport approximately 2.2-fold in the absence of aldosterone. These results suggest that aldosterone signals to the luminal Na(+) channel via multiple pathways and that K-Ras2A levels are limiting for a portion of the aldosterone-sensitive Na(+) transport.     

Selective cytotoxicity of a bicyclic Ras inhibitor in cancer cells expressing K-Ras

Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras^G13D, that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.     

Ras regulatory interactions: Novel targets for anti-cancer intervention?

Advances in the understanding of Ras oncoprotein function suggest novel points for anti-tumor intervention. First, upstream-acting guanine nucleotide exchange factors and SH2/SH3 domain-containing adaptor proteins that link Ras with growth factor receptor tyrosine kinases have recently been characterized. Second, work on downstream-acting Ras effector functions including the Ras GTPase-activating protein (p120^GAP) and the Raf kinase has revealed direct biochemical interactions that are functionally required for oncogenic Ras signalling. We summarize progress in these areas and discuss the potential for novel applications to anti-cancer chemotherapy.