Ras GTP Research Articles

Abstract PR02: Factors modulating RAF dimerization downstream of RAS – A mechanistic overview

Abstract The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. The activation of this pathway is tightly regulated with appropriate spatial and temporal signaling cues. The central step in activation of this pathway is the dimerization of RAF kinases. Recent advances in biochemical, enzymatic and structural characterization of various multiprotein complexes in this pathway provide insight into various modes of RAF activity modulation. Kinase RAF and its substrate MEK exist as a pre-formed complex prior to pathway activation. Multiple factors prevent MEK phosphorylation by RAF in this pre-formed complex, including phosphorylation of a Serine residue N-terminal to the kinase domain, which enables 14-3-3 to trap RAF as a monomer. One of our intriguing findings is that cellular ATP exerts a negative regulatory effect on RAF kinase and stabilizes the inactive conformation of RAF monomer. We characterize this negative regulatory effect of ATP of RAF kinase structurally by solving a RAF-ATP complex and enzymology. Upon membrane recruitment of RAF by RAS-GTP, dephosphorylation of the above-mentioned serine residue in RAF occurs by a multiprotein complex composed of phosphatase PP1C, a scaffolding protein SHOC2 and RAS-GTP. We solved the structure of this RAS-SHOC2-PP1C complex and performed a thorough enzymatic analysis of this complex for RAF dephosphorylation. Our results show that RAF specificity is determined by SHOC2 and RAS GTP is responsible for spatial localization both inactive RAF and SHOC2/PP1C to the membrane. This dephosphorylation results in RAF being stabilized as a dimer by 14-3-3, which now binds to phospho-serine residues C-terminal to the kinase domain (S729 in BRAF) in 2 RAF molecules. We solve a structure of the RAF-14-3-3 complex. 14-3-3 induced RAF dimerization increases activity of RAF ~500 fold. Further, oncogenic mutations in RAF and MEK occur basally in tumors, and additional mutations are induced upon pathway inhibitor treatment in the clinic, especially for recent KRAS-G12C treatments. These mutations include RAF mutations that either activate or inactivate the kinase activity of the mutated RAF, but nevertheless result in pathway activation. Our data suggests that these mutations appear to tilt the RAF monomer-dimer equilibrium towards RAF dimer. The molecular mechanism of how the MEK mutations active the pathway in poorly understood. Biochemical and structural characterization of these mutations occurring in MEK kinases suggests that the mechanism of activation is also likely functioning via promoting RAF dimerization. This RAF dimer promotion appears to mechanistically work by relieving the negative regulatory effect of RAF by ATP to result in the “just-right” amount of pathway activation to sustain tumor growth. A comprehensive understanding of the various factors modulating RAF dimerization in the context of normal cell and cancer cells that depend on this pathway can pave the way for more precise interventions for treatment of cancers that depend on the RAS pathway. Citation Format: Jawahar Sudhamsu, Nicholas Liau, Timothy Wendorff, Saeed Izadi, Luca Gerosa. Factors modulating RAF dimerization downstream of RAS – A mechanistic overview [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR02.

Mechanistic Insights into the Long-range Allosteric Regulation of KRAS Via Neurofibromatosis Type 1 (NF1) Scaffold Upon SPRED1 Loading

Allosteric regulation is the most direct and efficient way of regulating protein function, wherein proteins transmit the perturbations at one site to another distinct functional site. Deciphering the mechanism of allosteric regulation is of vital importance for the comprehension of both physiological and pathological events in vivo as well as the rational allosteric drug design. However, it remains challenging to elucidate dominant allosteric signal transduction pathways, especially for large and multi-component protein machineries where long-range allosteric regulation exits. One of the quintessential examples having long-range allosteric regulation is the ternary complex, SPRED1–RAS–neurofibromin type 1 (NF1, a RAS GTPase–activating protein), in which SPRED1 facilitates RAS–GTP hydrolysis by interacting with NF1 at a distal, allosteric site from the RAS binding site. To address the underlying mechanism, we performed extensive Gaussian accelerated molecular dynamics simulations and Markov state model analysis of KRAS–NF1 complex in the presence and absence of SPRED1. Our findings suggested that SPRED1 loading allosterically enhanced KRAS–NF1 binding, but hindered conformational transformation of the NF1 catalytic center for RAS hydrolysis. Moreover, we unveiled the possible allosteric pathways upon SPRED1 binding through difference contact network analysis. This study not only provided an in-depth mechanistic insight into the allosteric regulation of KRAS by SPRED1, but also shed light on the investigation of long-range allosteric regulation among complex macromolecular systems.

Structure of the MRAS–SHOC2–PP1C phosphatase complex

RAS–MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1–3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF–MEK1–14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS–SHOC2–PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF–RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies—developmental syndromes caused by aberrant MAPK pathway activation6–14—and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)–RAS-driven tumours15–18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS–SHOC2–PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS–GTP molecules recruit RAF–14-3-3 and SHOC2–PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein–protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS–MAPK signalling, therefore providing the structural basis for therapeutic interventions.

NMR Detection Methods for Profiling RAS Nucleotide Cycling.

RAS oncoproteins exhibit a switch-like behavior to drive diverse signaling cascades. In the active GTP-bound state, a conformational change occurs in these enzymes that enables interaction with downstream effectors. Nucleotide-dependent conformational exchange is easily detected with real-time NMR (RT-NMR) spectroscopy. RT-NMR has been firmly established as an effective assay to measure RAS oncoprotein nucleotide exchange and GTP hydrolysis kinetics and can further determine the regulatory activity of guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). It is now possible to multiplex these assays, allowing for the precise monitoring of activation states for mixtures of RAS oncoproteins or other RAS superfamily GTPases. Here, we describe the protocols necessary to express and purify isotopically labeled RAS and detail how to carry out an RT-NMR assay on a singular RAS protein or on a mixture of small GTPases.

Role of RASA1 in cancer: A review and update (Review).

Ras p21 protein activator 1 (RASA1) is a regulator of Ras GDP and GTP and is involved in numerous physiological processes such as angiogenesis, cell proliferation, and apoptosis. As a result, RASA1 also contributes to pathological processes in vascular diseases and tumour formation. This review focuses on the role of RASA1 in multiple tumours types in the lung, intestines, liver, and breast. Furthermore, we discuss the potential mechanisms of RASA1 and its downstream effects through Ras/RAF/MEK/ERK or Ras/PI3K/AKT signalling. Moreover, miRNAs are capable of regulating RASA1 and could be a novel targeted treatment strategy for tumours.

A subset of flavaglines inhibits KRAS nanoclustering and activation.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications.

A phase I, open-label, dose-escalation trial of BI 1701963 as monotherapy and in combination with trametinib in patients with KRAS mutated advanced or metastatic solid tumors.

TPS3651 Background: Activating mutations of KRAS drive many types of cancer. Activation of KRAS relies on guanine nucleotide exchange factors, such as SOS1, to mediate exchange of GDP for GTP. BI 1701963 is a small-molecule protein–protein interaction inhibitor that prevents the interaction between KRAS and SOS. Binding of BI 1701963 to the catalytic site of SOS1 inhibits binding of SOS1 to RAS–GDP, thereby hindering the exchange from RAS–GDP (inactive form) to RAS–GTP (active form). In preclinical studies this has been shown to lead to cytostasis in cancer cells addicted to KRAS signaling. Methods: NCT04111458 is a first-in-human trial of BI 1701963 in patients aged ≥18 years with tumors harboring KRAS mutations. Primary objectives are to determine the maximum tolerated dose (MTD) and recommended Phase II dose of BI 1701963 as monotherapy and in combination with trametinib, based on dose-limiting toxicities (DLTs). Secondary objectives are to evaluate safety, tolerability, pharmacokinetics/-dynamics and preliminary efficacy. The study will have two arms (mono- and combination therapy), and be divided into dose escalation (Part A), confirmation (Part B) and expansion (Part C, combination only) phases. Inclusion criteria include activating KRAS mutation, ≥1 evaluable lesion (RECIST 1.1), ECOG PS ≤1 and adequate organ function. Exclusion criteria include history of: RAS, MAPK or SOS1 targeting therapies; retinal vein occlusion; retinal pigment epithelial detachment; and decreased cardiac function. Parts B and C will be conducted in patients with advanced NSCLC. Treatment will continue until confirmed clinical benefit, defined toxicities, or withdrawal of consent. The primary endpoints are: Part A, the MTD, and the number of patients with DLTs during Cycle 1; Part B (monotherapy), the number of patients with DLTs; Part C, objective response (OR, RECIST 1.1). Starting doses of BI 1701963 in Part A will be 50 mg once daily (QD) orally (monotherapy) and 100 mg QD (combination, once proved safe as monotherapy) and will be escalated until the MTD is reached. The starting dose of trametinib will be 1 mg QD, escalated to the MTD or a max. of 2 mg QD. Dose cohorts will include ≥3 patients, with two therapeutic relevant doses (TRDs) established in each arm. In Part B, patients will be randomized to groups receiving one of the TRDs. If an OR is observed at a TRD in the combination arm, additional patients will be recruited into expansion cohorts receiving the relevant dose. As of Feb 11, 2020 three patients have been treated. Clinical trial information: NCT04111458 .

Abstract B53: Differential response of distinct KRAS mutants to SHP2 inhibition

Abstract Introduction: RAS is mutated in 30% of tumors and is still considered “undruggable.” Among RAS genes KRAS is the most frequently mutated, mostly at residues G12, G13 and Q61, impairing its intrinsic and GAP-mediated GTP hydrolysis capacity (Prior et al., Cancer Res 2012). Nevertheless, specific mutations are differently distributed in human cancers (G12 89%, G13 9%, Q61 1%). In particular, G12D (36%) is prevalent over G12C (14%) and G12V (23%). Distinct oncogenic mutations differently affect RAS GTP hydrolysis capacity (Lu et al., Sci Rep 2016; Hunter et al., Mol Cancer Research 2015), with G12C preserving the highest GTPase activity and Q61 mutants being the most compromised. This implies that some KRAS mutants could be sensitive to upstream regulation, as suggested by the in vivo sensitivity of KRAS mutant tumors to SHP2 inhibitors (Mainardi et al. and Ruess et al., Nat Med 2018; Fedele et al., Cancer Discov 2018). In order to finely predict the response to the SHP2 inhibitor SHP099 (Chen et al., Nature 2016) in KRAS mutant patients, we used a panel of isogenic Rasless murine embryonic fibroblasts (MEFs) (Esposito et al., Semin Cancer Biol 2018) expressing human wild-type or mutant (G13D, G12C, G12D, G12V or Q61R) KRAS genes, and studied their response to SHP099, either alone or in combination with the MEK inhibitor AZD6244. Materials and Method: The panel of isogenic RAS-dependent MEFs was provided by the NCI RAS Initiative. In vitro, RAS-GTP loading was evaluated and dose-response curves generated based on cell viability assays. For xenograft experiments, 5x106 cells were subcutaneously injected in NSG mice. After tumor establishment (200mm3) mice were treated either with vehicle, SHP099 75mg/kg, AZD6244 25 mg/Kg or a combination of the last two. Results and Discussion: In vitro, we found that when cultured in 3% FCS, the most GTPase-proficient KRAS mutants (G13D and G12C) are more sensitive to SHP099 as compared to the most impaired (G12D and Q61R). Response to SHP099 was accompanied by a reduction in RAS-GTP loading. Surprisingly, in vivo results using xenograft models showed no correlation between residual GTPase activity of the mutants and sensitivity to SHP099. Nevertheless, synergy between SHP2 and MEK inhibitors treatment was more pronounced in the GTPase-proficent mutants, suggesting a possible correlation between residual intrinsic GTP hydrolysis capacity of the mutants and their resistance to MEK inhibitors through feedback activation of receptor tyrosine kinases (Sun et al., Cell Rep 2014). Conclusion: Our data confirm single-agent activity of SHP099 against proliferation driven by a subset of KRAS mutants, and suggest that mutants with higher residual GTPase capacity could better benefit from combined SHP2 and MEK inhibition. Citation Format: Sara Mainardi, Astrid Bosma, Antonio Mulero-Sánchez, René Bernards. Differential response of distinct KRAS mutants to SHP2 inhibition [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B53.

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Phosphorylated Rho-GDP directly activates mTORC2 kinase towards AKT through dimerization with Ras-GTP to regulate cell migration.

mTORC2 plays critical roles in metabolism, cell survival, and actin cytoskeletal dynamics via phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae Dictyostelium cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho-GDP promotes AKT phosphorylation by assembling the supercomplex with Ras-GTP and mTORC2. This supercomplex formation is controlled by chemoattractant-induced phosphorylation of Rho-GDP at serine 192 by GSK-3. Furthermore, Rho-GDP rescued defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of serine 192. Thus, in contrast to the prevailing view that GDP-bound forms of G proteins are inactive, our study reveals that mTORC2-AKT signaling is activated by Rho-GDP.

Analysis of correlated mutations in Ras G-domain.

Ras GTPases are most prevalent proto-oncogenes in human cancer. Mutations in Ras remain untreatable more than three decades after the initial discovery. At the amino acid level, some residues under physical or functional constraints exhibit correlated mutations also known as coevolving/covariant residues. Revealing intra-molecular co-evolution between amino acid sites of proteins has become an emerging area of research as it enlightens the importance of variable regions. Here, I have identified and analyzed the coevolving residues in the Ras GTP binding domain (G-domain). The obtained covariant residue position data correlate well with the known experimental data on functionally important residues. Therefore, it is of interest to understand these residue co-variations for designing protein engineering experiments and target oncogenic Ras GTPases.

Abstract 5276: Phospholipase A/acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signal pathway

Abstract H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family that includes HREV107, Retinoid-inducible gene 1, and HRASLS. The HREV107 protein family contains an NC domain, with unknown function at the N-terminus, and a hydrophobic membrane-anchoring domain at the C-terminus. Previous study showed that the H-rev107 gene specifically expressed in H-ras resistant fibroblasts. However, the mode of action and the site of inhibition of H-rev107 are still unknown. Our results revealed that H-rev107 reduced the levels of activated RAS (RAS-GTP) or Elk1-mediated transactivation in epidermal growth factor stimulated HtTA cells that expressed H-RAS. Further, we found that H-rev107 reduced H-RAS palmitoylation determined by acyl-biotin exchange assay. Palmitoylation or S-acylation is the post-translational attachment of fatty acids to cysteine residues and continuous cycles of de- and reacylation reactions accounts for the specific subcellular distribution of RAS. H-rev107 was shown as a phospholipase A/Acyltransferase (PLA/AT) that involved in the phospholipid metabolism. We next determined whether H-rev107 affected H-RAS palmitoylation through deacylation/acylation by acyl-protein thioesterases (APT1) or PLA/AT using APT1 inhibitor (palmostatin B) or PLA/AT inhibitor (arachidonyl trifluoromethyl ketone, AACOCF3). Treating cells with AACOCF3 can increase H-rev107-induced acylated H-RAS suppression of HtTA cells. Also, AACOCF3 significantly increased the levels of activated RAS and Elk-mediated transactivation in H-rev107 expressed cells. However, treating cells with palmostatin B enhanced H-rev107-induced acylated H-RAS suppression in H-rev107 expressing cells. In conclusion, H-rev107 can inhibit H-RAS palmitoylation to reduce its GTP-form levels and further suppress the downstream signal pathway. Our study suggested that PLA/AT activity of H-rev107 might play an important role in H-rev107 mediated RAS suppression. Note: This abstract was not presented at the meeting. Citation Format: Fu-Ming Tsai, Chang-Chieh Wu, Chun-Hua Wang, Tzung-Chieh Tsai, Rong-Yaun Shyu, Shun-Yuan Jiang. Phospholipase A/acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signal pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5276. doi:10.1158/1538-7445.AM2014-5276

Unraveling the novel function of the DNA repair enzyme 8-oxoguanine-DNA glycosylase in activating key signaling pathways

Unraveling the novel function of the DNA repair enzyme 8-oxoguanine-DNA glycosylase in activating key signaling pathways

Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway

BackgroundH-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.ResultsOur results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.ConclusionsWe made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

Characterization of rare transforming KRAS mutations in sporadic colorectal cancer

KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P < 0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10–16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.

Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras-GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras-GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12-induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras-GTP to stimulate Ras-induced senescence in nontransformed human cells.

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P(i) by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10(5) compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10(5) compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.

Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction

The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA.

DHA alters EGFR spatiotemporal dynamics to suppress signal transduction

The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral in colon tumorigenesis. Lipid rafts play a role in controlling EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb lipid rafts. Here, we investigated the mechanistic link between EGFR function and DHA. DHA treatment of immortalized colonocytes and C57BL6 mouse colonic mucosa increased EGFR phosphorylation but paradoxically suppressed downstream activation of STAT3 and ERK1/2. DHA also suppressed cell proliferation in an EGFR‐dependent manner. The EGFR‐Ras‐ERK1/2 signaling cascade was assessed to identify the locus of the DHA‐induced disruption of signal transduction. Recruitment of Grb2 to EGFR, assessed using total internal reflective fluorescence (TIRF) microscopy, was increased by DHA treatment, whereas Ras GTP binding, a lipid raft‐dependent process, was suppressed by DHA treatment. Colocalization and membrane subfractionation experiments demonstrated that DHA reduced the localization of EGFR to lipid rafts. DHA further antagonized EGFR signaling capacity by increasing the rates of EGFR internalization and degradation. From these results, we conclude that DHA‐induced dissociation of EGFR from lipid rafts culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA.Grant Funding Source : NIH CA59034