Ras-like GTPase Research Articles

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.

The roles of ERAS during cell lineage specification of mouse early embryonic development.

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.

Rap1 GTPase is required for mouse lens epithelial maintenance and morphogenesis

Rap1, a Ras-like small GTPase, plays a crucial role in cell−matrix adhesive interactions, cell−cell junction formation, cell polarity and migration. The role of Rap1 in vertebrate organ development and tissue architecture, however, remains elusive. We addressed this question in a mouse lens model system using a conditional gene targeting approach. While individual germline deficiency of either Rap1a or Rap1b did not cause overt defects in mouse lens, conditional double deficiency (Rap1 cKO) prior to lens placode formation led to an ocular phenotype including microphthalmia and lens opacification in embryonic mice. The embryonic Rap1 cKO mouse lens exhibited striking defects including loss of E-cadherin- and ZO-1-based cell−cell junctions, disruption of paxillin and β1-integrin-based cell adhesive interactions along with abnormalities in cell shape and apical−basal polarity of epithelium. These epithelial changes were accompanied by increased levels of α-smooth muscle actin, vimentin and N-cadherin, and expression of transcriptional suppressors of E-cadherin (Snai1, Slug and Zeb2), and a mesenchymal metabolic protein (Dihydropyrimidine dehydrogenase). Additionally, while lens differentiation was not overtly affected, increased apoptosis and dysregulated cell cycle progression were noted in epithelium and fibers in Rap1 cKO mice. Collectively these observations uncover a requirement for Rap1 in maintenance of lens epithelial phenotype and morphogenesis.

Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells.

BackgroundCilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells.Methods and ResultsHuman aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis.ConclusionsThe PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis.

The mechanism and function of mitogen-activated protein kinase activation by ARF1

Mitogen-activated protein kinases (MAPK) can be activated by a number of biochemical pathways through distinct signaling molecules. We have recently revealed a novel function for the Ras-like small GTPase ADP-ribosylation factor 1 (ARF1) in mediating the activation of Raf1–MEK–ERK1/2 pathway by G protein-coupled receptors [Dong C, Li C and Wu G (2011) J Biol Chem 286, 43,361–43,369]. Here, we have further defined the underlying mechanism and the possible function of ARF1-mediated MAPK pathway. We demonstrated that the blockage of ARF1 activation and the disruption of ARF1 localization to the Golgi by mutating Thr48, a highly conserved residue involved in the exchange of GDP for GTP, and the myristoylation site Gly2 abolished ARF1's ability to activate ERK1/2. In addition, treatment with Golgi structure disrupting agents markedly attenuated ARF1-mediated ERK1/2 activation. Furthermore, ARF1 significantly promoted cell proliferation. More interestingly, ARF1 activated 90kDa ribosomal S6 kinase 1 (RSK1) without influencing Elk-1 activation and ERK2 translocation to the nuclei. These data demonstrate that, once activated, ARF1 activates the MAPK pathway likely using the Golgi as a main platform, which in turn activates the cytoplasmic RSK1, leading to cell proliferation.

The small GTPase MoYpt7 is required for membrane fusion in autophagy and pathogenicity of Magnaporthe oryzae.

Rab GTPases are required for vesicle-vacuolar fusion during vacuolar biogenesis in fungi. To date, little is known about the biological functions of the Rab small GTPase components in Magnaporthe oryzae. In this study, we investigated MoYpt7 of M. oryzae, a homologue of the small Ras-like GTPase Ypt7 in Saccharomyces cerevisiae. Cellular localization assays showed that MoYpt7 was predominantly localized to vacuolar membranes. Using a targeted gene disruption strategy, a ΔMoYPT7 mutant was generated that exhibited defects in mycelial growth and production of conidia. The conidia of the ΔMoYPT7 mutant were malformed and defective in the formation of appressoria. Consequently, the ΔMoYPT7 mutant failed to cause disease in rice and barley. Furthermore, the ΔMoYPT7 mutant showed impairment in autophagy, breached cell wall integrity, and higher sensitivity to both calcium and heavy metal stress. Transformants constitutively expressing an active MoYPT7 allele (MoYPT7-CA, Gln67Leu) exhibited distinct phenotypes from the ΔMoYPT7 mutant. Expression of MoYPT7-CA in MoYpt7 reduced pathogenicity and produced more appressoria-forming single-septum conidia. These results indicate that MoYPT7 is required for fungal morphogenesis, vacuole fusion, autophagy, stress resistance and pathogenicity in M. oryzae.

Leukocyte transcellular diapedesis: Rap1b is in control

The neutrophil transmigration across the blood endothelial cell barrier represents the prerequisite step of innate inflammation. It is well known that neutrophils cross the endothelial barrier by transmigrating at the endothelial cell junction (‘paracellular’). However, in vivo and in vitro evidence have clearly demonstrated occurrence of an alternate mode of migration directly through the endothelial cell body (‘transcellular’). Despite our knowledge on mechanisms of transendothelial migration, it remains unclear which factors determine distinct modes of migration. We recently found that the Ras-like Rap1b GTPase limits neutrophil transcellular migration. Rap1b restrains transcellular migration by suppressing Akt-driven invasive protrusions while leaving the paracellular route unaffected. Furthermore, Rap1b limits neutrophil tissue infiltration in mice and prevents hyper susceptibility to endotoxin shock. These findings uncover a novel role for Rap1b in neutrophil migration and inflammation. Importantly, they offer emerging evidences that paracellular and transcellular migration of neutrophils are regulated by separate mechanisms. Here, we discuss the mechanisms of neutrophil transmigration and their clinical importance for vascular integrity and innate inflammation.

Exploring bacterial organelle interactomes: a model of the protein-protein interaction network in the Pdu microcompartment.

Bacterial microcompartments (MCPs) are protein-bound organelles that carry out diverse metabolic pathways in a wide range of bacteria. These supramolecular assemblies consist of a thin outer protein shell, reminiscent of a viral capsid, which encapsulates sequentially acting enzymes. The most complex MCP elucidated so far is the propanediol utilizing (Pdu) microcompartment. It contains the reactions for degrading 1,2-propanediol. While several experimental studies on the Pdu system have provided hints about its organization, a clear picture of how all the individual components interact has not emerged yet. Here we use co-evolution-based methods, involving pairwise comparisons of protein phylogenetic trees, to predict the protein-protein interaction (PPI) network governing the assembly of the Pdu MCP. We propose a model of the Pdu interactome, from which selected PPIs are further inspected via computational docking simulations. We find that shell protein PduA is able to serve as a “universal hub” for targeting an array of enzymes presenting special N-terminal extensions, namely PduC, D, E, L and P. The varied N-terminal peptides are predicted to bind in the same cleft on the presumptive luminal face of the PduA hexamer. We also propose that PduV, a protein of unknown function with remote homology to the Ras-like GTPase superfamily, is likely to localize outside the MCP, interacting with the protruding β-barrel of the hexameric PduU shell protein. Preliminary experiments involving a bacterial two-hybrid assay are presented that corroborate the existence of a PduU-PduV interaction. This first systematic computational study aimed at characterizing the interactome of a bacterial microcompartment provides fresh insight into the organization of the Pdu MCP.

Involvement of RalB in the effect of geranylgeranyltransferase I on glioma cell migration and invasion.

The present study aimed at investigating the role and potential mechanism of geranylgeranyltransferase I (GGTase-I) on glioma cell migration and invasion. Wound-healing assay and transwell assay were performed to study whether GGTase-I and Ras-like GTPase B (RalB) have effect on glioma cell migration and invasion. We found that knockdown of GGTase-I or RalB both significantly decreased the migratory and invasive abilities of glioma cells. GGTase-I down-regulation suppressed RalB membrane association. Moreover, down-regulation of RalB partially abolished the effect of GGTβ over-expression-induced glioma cell migration and invasion increase. These findings suggest that RalB might be one of the targets for facilitating the invasive phenotype of malignant gliomas induced by GGTase-I.

Conformational Transitions in Switch Regions of the Ras-Like GTPase Rab1B Studied by Free Energy Simulations

The family of small Ras-like GTPases plays a pivotal role in the coordination of cell signaling by acting as binary protein switches. The switching mechanism is triggered by the hydrolysis of co-factor GTP to GDP which induces a transition in conformation and dynamics of two functional regions designated as switch I and switch II located in the proximity of the co-factor binding site. The switch I and switch II regions typically adopt an ordered and well defined structure only in the presence of bound GTP and unfold in the presence of GDP. The conformational equilibrium of switch I and switch II regions can be further influenced by post-translational modifications. We employed umbrella sampling free energy simulations to systematically investigate the switch I and switch II conformational transitions in the Ras-like GTPase Rab1b in the presence of bound GTP or GDP and different posttranslational modifications. The free energy change was recorded along a collective coordinate composed of a network of predefined inter-atomic distances (dRMSD) coupled with Hamiltonian replica exchange simulations. In agreement with experiment the free energy simulations predicted a lower free energy barrier of unfolding switch I and switch II regions in the presence of GDP compared to GTP and are also compatible with the experimentally observed effects of posttranslational modifications. An energetic analysis indicates that electrostatic interactions play a decisive role for the conformational equilibrium. It was also possible to determine the order of structural transitions in the switch I and II regions upon induced unfolding. The results can have important implications for understanding the mechanism of signal transduction by GTPases.

Rab-NANOPS: FRET biosensors for Rab membrane nanoclustering and prenylation detection in mammalian cells.

Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane anchorage, subcellular distribution, and of the nanoscale organization of Rab-GTPases in mammalian cell lines. This assay is high-throughput amenable and can therefore be utilized in chemical-genomic and drug discovery efforts.

Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways.

Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis.

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.

Identification and function of 11 Rab GTPases in giant freshwater prawn Macrobrachium rosenbergii

Rab GTPases, members of the Ras-like GTPase superfamily, are central elements in endocytic membrane trafficking. However, little is known of the Rab genes in the giant freshwater prawn Macrobrachium rosenbergii. In this study, 11 Rab genes were identified from M. rosenbergii. All MrRabs have a RAB domain. Phylogenetic analysis showed that these 11 MrRabs were divided into different groups. The MrRab genes were ubiquitously expressed in heart, hemocytes, hepatopancreas, gills, stomach, and intestines. Real-time polymerase chain reaction revealed that the MrRab genes were significantly upregulated by white spot syndrome virus (WSSV) in the prawns, indicating that MrRabs might play an important role in innate immune response against WSSV. Moreover, after challenge with Vibrio parahaemolyticus, the expression levels of all MrRabs in the hepatopancreas were also upregulated, which might indicated the involvement of MrRabs in prawns antibacterial immunity. In all, these preliminary results showed that MrRabs were involved in innate immunity of M. rosenbergii.

Abstract A08: Mechanistic dissection of Ral GTPase signaling in driving KRAS-dependent pancreatic cancer growth

Abstract The high frequency (95%) of KRAS mutations in pancreatic ductal adenocarcinomas (PDAC), together with strong evidence for the KRAS dependency of PDAC growth, makes the K-Ras oncoprotein an attractive therapeutic target. However, to date the development of direct inhibitors of K-Ras has not been successful, with most efforts focused on targeting components of K-Ras downstream effector signaling. While the pharmaceutical industry has centered on inhibitors of the Raf or PI3K effector signaling pathways, there is substantial evidence that K-Ras-dependent cancer growth must rely on the activities of other effector pathways. Of those pathways, the Ral guanine nucleotide exchange factor RalGEF) effector pathway, leading to activation of the Ras-like (Ral) small GTPases is well-validated to drive PDAC growth. Interestingly, while the two Ral isoforms share significant sequence identity (82%) as well as the same set of effectors, our studies found that they have strikingly different roles in PDAC growth. While RalA but not RalB is necessary for PDAC anchorage-independent growth in vitro and tumorigenic growth in vivo, RalB is the critical driver of PDAC invasion in vitro and metastasis in vivo. The mechanistic basis for their different biological roles in cancer remains to be elucidated. We hypothesize that their divergent C-terminal sequences and the posttranslational modifications that they signal for result in distinct subcellular localizations that then contribute to Ral isoform-selective effector interactions that promote distinct biological consequences in PDAC. To evaluate our hypothesis, we initiated studies to determine the effector(s) that mediates RalA-dependent anchorage-independent growth and tumorigenesis in vivo, the effector(s) that mediates RalB-dependent invasion and metastasis in vivo; and the role of the C-terminal hypervariable domain in causing distinct RalA- and RalB-dependent phenotypes and effector utilization. We found that both RalA and RalB subcellular localization is dependent on the C-terminal CAAX tetrapeptide signaled modifications by Rce1 and ICMT. We also determined that RalB preferentially activated mTORC1 to regulate pancreatic tumor cell Matrigel migration, through the Sec5 effector and regulation of exocyst function. Citation Format: Leanna R. Gentry, Timothy D. Martin, David J. Reiner, Channing J. Der. Mechanistic dissection of Ral GTPase signaling in driving KRAS-dependent pancreatic cancer growth. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A08. doi: 10.1158/1557-3125.RASONC14-A08

Ral-lying around Ras

A University of Colorado–led team has demonstrated anticancer efficacy for new small molecule inhibitors of Ral proteins—a family of Ras-like GTPases often overlooked in favor of their Ras cousins.

Abstract 1781: Identification of focal adhesion and actin cytoskeleton regulation family genes as druggable target for gastric cancer

Abstract Gastric cancer(GC) is the 4th most common malignancy in the world and the 2nd most common cause of cancer related death worldwide (1). The known causes of GC are diet, hygiene, Helicobacter pylori, Epstein-Barr virus and hereditary factors. Other genetic causes of GC are known to be heterogenic, partially due to differences in ethnic/genetic background. Surgical removal of the tumor is the first-line treatment. Targeted therapy agents and chemotherapeutic agents that show survival advantage in other cancer types are being evaluated in GC(2,3), which only target few known oncogenes. Targeted therapy for GC is still an unmet need, and in light of personalized medicine, identifying genes specific to GC is critical for its treatment. From our NGS data, we have discovered protein family members involved in focal adhesion and actin cytoskeleton regulation pathways, which are involved in mitosis and metastasis. We developed a subpathway network analysis method called PATHOME(unpublished), to identify pathways related to GC. For the first stage, two datasets (84 Korean GC: GSE13861 and 56 Japanese GC:GSE15081)(4,5). Two datasets (25 Korean GC: GSE36968 and 160 Chinese GC:GSE27342)(6,7,8) were used as validation sets. We identified top 10 common pathways. We have then categorized genes within these pathways according their functional category and druggability, either as drug transporter or druggable target. As a result, we identified genes involved in the focal adhesion and regulation of actin cytoskeleton pathways and cell junction, all critical for cell division and metastasis of tumor cells. Genes G1-9 are tight junction genes, and G10-20 are protein family group related to Ras-like GTP-binding protein, G10 - 20. G10, 11 and 12 are ras-like GTPases, and G13-20 are interacting proteins or downstream proteins. We then compared differential expression of these genes between the Asian and Caucasian dataset. G1-9 show co-regulation pattern among the two ethnic group. G9 in particular, is normally expressed in the stomach, and is downregulated (p=0.0045) in GC tumor samples. In contrast, G10 is upregulated in the Korean dataset and is shown to be upregulated in Japanese and Chinese GC sample (public) as well(9,10), but not in our Caucasian dataset(unpublished). The TCGA stomach cancer provisional data via cBioPortal(11) shows that most of the G10 alterations in GC cases are mutations (9% of total cases) with 2 known cases of downregulation. It is interesting that most of the G10 alterations were found in Caucasian patients (11/18 cases). In our study, we have identified genes that are significant in GC and are druggable targets/drug transporters. It is significant that the pathways we identified are detected in both ethnic groups, with different expression pattern. This finding is critical for personalized treatment and drug development for GC. Citation Format: Hae Ryung Chang, Seungyoon Nam, Myeong-Cherl Kook, Hee Seo Park, Hae Rim Jung, Youme Gim, Han Liang, Garth Powis, Yon Hui Kim. Identification of focal adhesion and actin cytoskeleton regulation family genes as druggable target for gastric cancer. [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 1781. doi:10.1158/1538-7445.AM2014-1781

Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors.

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-β) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-β-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-β-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-β receptor II (TβRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF-β1 stimulation. Our study demonstrates a requirement of VASP for TGF-β-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver.

Discovery and characterization of small molecules that target the GTPase Ral.

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.

Regulation of cell polarity in bacteria.

Bacteria are polarized cells with many asymmetrically localized proteins that are regulated temporally and spatially. This spatiotemporal dynamics is critical for several fundamental cellular processes including growth, division, cell cycle regulation, chromosome segregation, differentiation, and motility. Therefore, understanding how proteins find their correct location at the right time is crucial for elucidating bacterial cell function. Despite the diversity of proteins displaying spatiotemporal dynamics, general principles for the dynamic regulation of protein localization to the cell poles and the midcell are emerging. These principles include diffusion-capture, self-assembling polymer-forming landmark proteins, nonpolymer forming landmark proteins, matrix-dependent self-organizing ParA/MinD ATPases, and small Ras-like GTPases.