GTP-bound State Research Articles

Comparative Structural Dynamic Analysis of G Proteins

G proteins are molecular switches that participate in a multitude of essential cellular processes ranging from movement and division to differentiation and neuronal activity. These ubiquitous GTPases hydrolyze GTP to GDP with associated conformational changes that modulate affinity for specific protein binding partners. There are two major G protein superfamilies: small Ras-like GTPases and larger heterotrimeric G proteins. Although they contain an analogous nucleotide binding domain, the detailed mechanisms by which these structurally and functionally diverse superfamilies operate remain unclear. Here we compare and contrast the structural dynamic mechanisms of Ras and transducin using conventional molecular dynamics (MD). In particular, we analyze the cross-correlations of atomic displacements in both the GTP-bound and GDP-bound states together with detailed network analysis of residues that determine their unique functions. Our analysis reveals consistent dynamic subdomain structures in the catalytic domain of both Ras and transducin, indicating that the two proteins use a similar framework for allostery. However, nucleotide dependent transitions of inter-subdomain couplings are found to be distinct between these two proteins, which may define their specific functions. In addition, residues mediating the coupling of distal functional sites in Ras are identified and compared with the allosteric residues from G[alpha]i discovered in our previous work. Our studies extend current understanding of G protein allosteric mechanisms and provide an exclusive insight into the evolution of G protein superfamilies.

Ras Conformational Ensembles, Allostery, and Signaling.

Ras proteins are classical members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. In this review, we focus on data that have accumulated over the past few years pertaining to the conformational ensembles and the allosteric regulation of Ras proteins and their interpretation from our conformational landscape standpoint. The Ras ensemble embodies all states, including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states serving as a reservoir for emerging functions. The ensemble is shifted by distinct mutational events, cofactors, post-translational modifications, and different membrane compositions. A better understanding of Ras biology can contribute to therapeutic strategies.

REI/SH3BP5 protein family: New GEFs for Rab11

The Rab family of proteins are key small GTPases that play essential roles in membrane trafficking. The Rab proteins cycle between an inactive GDP-bound state and an active GTP-bound state. This GD...

Analysis of Rho-GTPase Activity During Budding Yeast Cytokinesis.

Rho-type small GTPases are involved in cytokinesis in various organisms, but their precise roles and regulation remain unclear. Rho proteins function as molecular switches by cycling between the active GTP-bound and inactive GDP-bound states; the GTP-bound proteins in turn interact with their downstream effectors to transmit the signal. Biochemical assays using Rho-binding domains of effector proteins have been used to specifically pull down GTP-bound Rho proteins from cell extracts. Here, we describe the application of such a method in combination with cell-cycle synchronization in the budding yeast Saccharomyces cerevisiae; this approach allows dissection of the activity of Rho1 at different stages of cytokinesis. We also present data showing the importance of caution in interpreting such biochemical data and of comparing to the results obtained with other approaches where possible. The principle of this protocol is also applicable to analyses of other Rho-type GTPases and cell-cycle events.

RAB21 Activity Assay Using GST-fused APPL1.

The Rab family of small GTPases are essential regulators of membrane trafficking events. As with other small GTPase families, Rab GTPases cycle between an inactive GDP-bound state and an active GTP-bound state. Guanine nucleotide exchange factors (GEFs) promote Rab activation with the exchange of bound GDP for GTP, while GTPase-activating proteins (GAPs) regulate Rab inactivation with GTP hydrolysis. Numerous methods have been established to monitor the activation status of Rab GTPases. Of those, FRET-based methods are used to identify when and where a Rab GTPase is activated in cells. Unfortunately, the generation of such probes is complex, and only a limited number of Rabs have been probed this way. Biochemical purification of activated Rabs from cell or tissue extracts is easily achievable through the use of a known Rab effector domain to pull down a specific GTP-bound Rab form. Although this method is not ideal for detailed subcellular localization, it can offer temporal resolution of Rab activity. The identification of a growing number of specific effectors now allows tests for activation levels of many Rab GTPases in specific conditions. Here, we described an affinity purification approach using GST fused APPL1 (a known RAB21 effector) to test RAB21 activation in mammalian cells. This method was successfully used to assay changes in RAB21 activation status under nutrient rich versus starved conditions and to test the requirement of the MTMR13 RAB21 GEF in this process.

The higher level of complexity of K-Ras4B activation at the membrane.

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane.

Disordered Regions Flanking Ordered Domains Modulate Signaling Transduction

Signals generated by cell surface receptors are transduced and relayed to downstream intracellular signaling pathways typically through proteins in which folded domains are flanked by regions that are unstructured or only partially structured. Such signaling systems are often multidomain proteins in which disordered linkers provide an effective means to covalently tether and colocalize globular modules (1). However, in several regulatory proteins, linkers have been reported to serve not only as passive covalent tethers, but also as active modulators of the function elicited by adjacent domains (2–4).

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.

Inhibition of the regulator of G protein signalling RGS4 in the spinal cord decreases neuropathic hyperalgesia and restores cannabinoid CB1 receptor signalling.

Regulators of G protein signalling (RGS) are major determinants of metabotropic receptor activity, reducing the lifespan of the GTP-bound state of G proteins. Because the reduced potency of analgesic agents in neuropathic pain may reflect alterations in RGS, we assessed the effects of CCG 63802, a specific RGS4 inhibitor, on pain hypersensitivity and signalling through cannabinoid receptors, in a model of neuropathic pain. The partial sciatic nerve ligation (PSNL) model in male Sprague Dawley rats was used to measure paw withdrawal thresholds to mechanical (von Frey hairs) or thermal (Hargreaves method) stimuli, during and after intrathecal injection of CCG 63802. HEK293 cells expressing CB1 receptors and conditional expression of RGS4 were used to correlate cAMP production and ERK phosphorylation with receptor activation and RGS4 action. Treatment of PSNL rats with CCG 63802, twice daily for 7 days after nerve injury, attenuated thermal hyperalgesia during treatment. Spinal levels of anandamide were higher in PSNL animals, irrespective of the treatment. Although expression of CB1 receptors was unaffected, HU210-induced CB1 receptor signalling was inhibited in PSNL rats and restored after intrathecal CCG 63802. In transfected HEK cells expressing CB1 receptors and RGS4, inhibition of cAMP production, a downstream effect of CB1 receptor signalling, was blunted after RGS4 overexpression. RGS4 expression also attenuated the CB1 receptor-controlled activation of ERK1/2. Inhibition of spinal RGS4 restored endogenous analgesic signalling pathways and mitigated neuropathic pain. Signalling through CB1 receptors may be involved in this beneficial effect.

Novel topography of the Rab11-effector interaction network within a ciliary membrane targeting complex

Small GTPases function as universal molecular switches due to the nucleotide dependent conformational changes of their switch regions that allow interacting proteins to discriminate between the active GTP-bound and the inactive GDP-bound states. Guanine nucleotide exchange factors (GEFs) recognize the inactive GDP-bound conformation whereas GTPase activating proteins (GAPs), and the GTPase effectors recognize the active GTP-bound state. Small GTPases are linked to each other through regulatory and effector proteins into functional networks that regulate intracellular membrane traffic through diverse mechanisms that include GEF and GAP cascades, GEF-effector interactions, common effectors and positive feedback loops linking interacting proteins. As more structural and functional information is becoming available, new types of interactions between regulatory proteins, and new mechanisms by which GTPases are networked to control membrane traffic are being revealed. This review will focus on the structure and function of the novel Rab11-FIP3-Rabin8 dual effector complex and its implications for the targeting of sensory receptors to primary cilia, dysfunction of which causes cilia defects underlying human diseases and disorders know as ciliopathies.

Structure and Switch Cycle of SRβ as Ancestral Eukaryotic GTPase Associated with Secretory Membranes

G proteins of the Ras-family of small GTPases trace the evolution of eukaryotes. The earliest branching involves the closely related Arf, Sar1, and SRβ GTPases associated with secretory membranes. SRβ is an integral membrane component of the signal recognition particle (SRP) receptor that targets ribosome-nascent chain complexes to the ER. How SRβ integrates into the regulation of SRP-dependent membrane protein biogenesis is not known. Here we show that SRβ-GTP interacts with ribosomes only in presence of SRα and present crystal structures of SRβ in complex with the SRX domain of SRα in the GTP-bound state at 3.2 Å, and of GDP- and GDP · Mg(2+)-bound SRβ at 1.9 Å and 2.4 Å, respectively. We define the GTPase switch cycle of SRβ and identify specific differences to the Arf and Sar1 families with implications for GTPase regulation. Our data allow a better integration of SRβ into the scheme of protein targeting.

Biochemical And Tumorigenic Effects Of Redox Modification Of Ras-G12c By Nitric Oxide

The Ras family of small GTPases cycle between an inactive, GDP-bound state and an active, GTP-bound state. When bound to GTP, Ras engages and activates a number of effectors that mediate proliferative and survival signals. Ras is mutated in over 30% of human cancers, usually at codons 12, 13, or 61, to remain in this active, GTP-bound state, which promotes tumorigenesis. One of these oncogenic mutations that commonly occurs in lung cancer is G12C. Recently, it was shown that alkylating agents that react with the thiol functional group of this mutant amino acid can inactivate oncogenic RasG12C. Given that Cys12 of RasG12C is accessible to thiol alkylating agents and forms interactions within the electrostatic phosphoryl-binding loop of Ras, we postulated that Cys12 may possess an altered pKa, potentially allowing this residue to be modified by NO and other cellular oxidants. We conducted several biochemical analyses to determine whether nitrosylation of RasG12C alters its activity and structure in vitro. We also determined the biological effects of increasing NO production on the tumorigenic growth of cells transformed by RasG12C. We found that Cys12 has a depressed pKa of 7.4, which increases the susceptibility of the thiol to modification by oxidation or nitrosylation at physiological pH. We also found that coexpressing active eNOSS1177D and RasG12C accelerated tumorigenic growth of human and murine cell line xenografts. Modification of Cys12 in mutant, oncogenic RasG12C may promote its tumorigenic activity.

Abstract LB-058: Novel interactions of the RAS oncoprotein

Abstract Approximately 30% of all cancers harbor activating mutations in the RAS family of small GTPase proteins, making it one of the most common oncogenic aberrations in humans. Normal RAS proteins (H, K or N-RAS) localize to the inner cell membrane and transduce extracellular growth signals by cycling between an “active” GTP-bound state and “inactive” GDP-bound state, through interactions with various “GTPase activating proteins” (GAPs) that promote RAS mediated GTP hydrolysis. Oncogenic mutants of RAS lose their catalytic activity or association with the GAP proteins, resulting in constitutively active GTP-bound state that signals through direct interactions with effector kinases like RAF and PI3K and activate the MEK/ERK and/or Akt, leading to activation of hallmark cancer pathways including growth factor independence, uncontrolled cell proliferation, evasion of apoptosis and immune responses, increased metabolism as well as metastases. Although RAS is the most frequently mutated gene driving multifarious pathways of oncogenesis, our knowledge of protein interactions involving RAS proteins have been largely limited to RAS binding domains in RAF/PI3K/RalGDS. Targeting mutant RAS proteins or its direct effectors, or pathways activated by RAS effectors remains a challenging endeavor for treating RAS driven cancers. Towards the goal of a thorough understanding of RAS biology through a comprehensive identification of its interactors, we performed IP-Mass Spectrometric analysis of pan-RAS immunoprecipitates from multiple cell lines. Interestingly in our experiments, apart from the well-known interactor RAF, we found evidence of several novel RAS interacting proteins, including many with DNA and RNA binding motifs. Our study validates these findings through cell-free protein interaction analyses and explores the possible biological effects of these novel RAS interactions in mutant KRAS driven cellular transformation. Note: This abstract was not presented at the meeting. Citation Format: Sunita Shankar, Rohit Malik, Vishal Kothari, Yasuyuki Hosono, Sethuramasundaram Pitchiaya, Shanker Kalyana-Sundaram, Anastasia Yocum, June Escara-Wilke, Harika Gundlapalli, Krishnapriya Chinnaswamy, Matthew Shuler, Anton Poliakov, Xiaoju Wang, Vishalakshi Krishnan, Yasmine White, Ari Firestone, Xuhong Cao, Saravana M. Dhanasekaran, Jeanne Stuckey, Gideon Bollag, Kevin Shannon, Nils G. Walter, Chandan Kumar-Sinha, Arul Chinnaiyan. Novel interactions of the RAS oncoprotein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-058. doi:10.1158/1538-7445.AM2015-LB-058

Abstract LB-141: Specific and potent silencing of K-Ras by asymmetric silencing RNA (aiRNA) reveals addiction of cancer stem cells to mutant K-Ras amplification

Abstract K-Ras, the first oncogene identified in human cancer, is mutated in about 30% of human solid tumors. K-Ras protein, a small membrane-bound GTP-binding protein, acts as a molecular switch to transduce cell proliferation signals. Activating mutations of K-Ras lock the Ras protein into the hyper-active GTP-bound state, resulting in the activation of numerous signaling pathways that control cell survival and proliferation. Ras is also an important oncoprotein in many cancers where it is not mutated since Ras can be functionally activated through aberrant activation of other signal transduction elements. Activated K-Ras proteins are, therefore, found in a large proportion of all human cancers, and occupy a central position of interest. Hypermalignant cancer cells, termed cancer stem cells (CSCs), that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties. These stemness-high cancer cells are hypothesized to be fundamentally responsible for cancer metastasis and relapse. Furthermore, a number of stemness genes, such as beta-catenin, nanog, Sox2, Oct3/4 have been implicated in cancer cell stemness. The role of oncogenes such as K-Ras in cancer cell stemness, however, is not clear. To elucidate the role of K-Ras in the maintenance of cancer cell stemness, we employed asymmetric silencing RNA technology (aiRNA) which is able to silence target genes with high potency and precision. Moreover, aiRNA technology can be readily applied to CSCs. Here we report, to our surprise, that CSCs are not simply addicted to activating mutations of K-Ras, or activation of downstream regulators of the Ras pathway. However, CSCs with amplified mutant K-Ras are highly sensitive to K-Ras silencing. Moreover, the DNA copy number of the mutant K-Ras directly predicts the sensitivity of CSCs to K-Ras silencing. Our studies suggest that amplified mutated K-Ras is required for the maintenance of malignancy and cancer cell stemness, which may have significant implications for understanding the connections between oncogenes and cancer cell stemness, and for developing cancer stem cell inhibitors. Citation Format: Jun Oishi, Hiroki Umehara, Nithya Jesuraj, Jelena Barbulovic, Xiangao Sun, Chiang J. Li. Specific and potent silencing of K-Ras by asymmetric silencing RNA (aiRNA) reveals addiction of cancer stem cells to mutant K-Ras amplification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2015-LB-141

Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES) for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo.

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

Abstract 200: Vascular Stem Cell Migration in Response to Monocyte Chemoattractant Protein-1 Promotes Neointima Lesion Formation

Introduction: While vascular smooth muscle cells are traditionally known for their role in neointima formation, recent studies show that a population of Sca-1 + stem/progenitor cells within the adventitia of vessel walls can also contribute to restenosis formation. To date, their migratory properties and motility in response to fundamental micro-environmental stimuli within the vasculature remain unknown. The occurrence of adventitial inflammation in injured vessels or during atherosclerosis is known to mount an increased secretion of the chemokine, monocyte chemoattractant protein-1 (MCP-1), by cells such as macrophages, endothelial and smooth muscle cells. Hypothesis: We hypothesize that the MCP-1 plays an important role on the mobilization of vascular progenitor cells. Methods and Results: Vascular progenitor cells were derived from vein graft and isolated with an anti-Sca-1 antibody microbead system. Using transwell assays, we found that recombinant MCP-1 significantly increased progenitor cell migration. The knockdown of its receptor expression, CCR2, using lentiviral shRNA significantly inhibited MCP-1 induced migration. Mechanistically, cytoskeleton related GTPase rac1 and cdc42 in progenitor cells were activated (in the GTP-bound state) by MCP-1. Furthermore, we showed that the ERK 1/2 pathway was also involved in MCP-1-mediated migration. Consistently, the migration of progenitor cells was significantly decreased following treatment with a MAPKK inhibitor. Using a femoral artery wire injury mouse model, we found that the seeding of Sca-1 + progenitors on the outer layers of the adventitia markedly enhanced neointima formation after 1 week, whereas vessels without progenitor cell seeding did not form neointima. Neointima formation in Sca-1 + progenitors-seeded vessels were found inhibited in mice that lacked functional MCP-1 (n≥8 per group). Conclusion: In conclusion, MCP-1 can induce adventitia-derived Sca-1 + progenitor cell migration by the activation of GTP-rac1/cdc42 and ERK 1/2 signalling pathway via CCR2. The lack of functional MCP-1 inhibited progenitor cell migration and neointima formation in vivo .

Differential dynamics of RAS isoforms in GDP- and GTP-bound states.

RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP- (total of 3.06 μs) and GTP-bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25-40) and SwitchII (Residues 59-75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP-bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild-type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF-assisted nucleotide exchange.

K-Ras, Intestinal Homeostasis and Colon Cancer

Activating Ras mutations, present in about 20% of human cancers, compromise the GTPase activity of Ras and therefore trigger accumulation of Ras in the GTP-bound state. Among the three family members, K-Ras, H-Ras and N-Ras, K-Ras is the most frequently mutated gene, with 30-50% of colon cancer patients harboring activating K-Ras mutations. Oncogenic mutations of K-Ras have been found at codons 12, 13, 61 and 146. Activation of Ras triggers constitutive activation of signaling pathways, including the MAPK and AKT pathways, which allows tumor cells to proliferate in the absence of growth factors and increases their survival. In addition, activated Ras triggers inflammation and thus promotes tumor progression in a cell non-autonomous manner. The presence of K-Ras mutations not only has prognostic value, but it also predicts the responsiveness of colon cancer patients to inhibitors of EGFR signaling.

Pan-PI-3 kinase inhibitor SF1126 shows antitumor and antiangiogenic activity in renal cell carcinoma.

SF1126 is a vascular-targeted pan-PI-3K inhibitor prodrug with antitumor and antiangiogenic activity and has completed phase I clinical trial in solid tumors and B-cell malignancies. In this study, we investigated the effect of SF1126 on hypoxic HIF-1α/HIF-2α stability as well as on antitumor and/or antiangiogenic activity in renal cell carcinoma (RCC) models in vitro and in vivo. The effect of SF1126 on hypoxic HIF-1α/HIF-2α protein stability, antitumor and antiangiogenic activity was studied on VHL-null (786-0) and VHL-WT (Caki) RCC cells. Our data demonstrate that SF1126 treatment abrogates the stabilization of HIF-2α in 786-0 (VHL-mutated) RCC cell line under normoxic and hypoxic conditions. Similarly, hypoxic stabilization of HIF-1α and its activity were also suppressed following SF1126 treatment in Caki cell line (VHL-WT). Herein, we provide mechanistic evidence that HIF-2α can be degraded in cytoplasm under hypoxic conditions via the 26S proteasome and that MDM2 is the E3 ligase which induces the hypoxic degradation of HIF-2α in PI-3K-dependent manner in VHL-deficient RCC cells. Moreover, SF1126 administered to RCC-xenografted mice at 25mg/kg/dose subcutaneously three times per week for 3weeks results in marked inhibition of tumor growth (>90% inhibition) (P<0.05). Consistent with SF1126 treatment's effects on HIF-1α/HIF-2α, microvessel density analysis of Caki and 786-0 tumor tissues demonstrated that SF1126 has potent antiangiogenic activity in vivo. Finally, SF1126 caused a profound inhibition of integrin-mediated migration and blocked the integrin-induced conversion of GDP-Rac1 to its GTP-bound active state. These results validate the in vivo efficacy of SF1126 as a clinically viable antiangiogenic, pan-PI-3K inhibitor prodrug for phase II clinical trials in the treatment of RCC.