Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 45
Filter
Add more filters










Publication year range
1.
Int Immunopharmacol ; 83: 106454, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32259700

ABSTRACT

Lung adenocarcinoma (LUAD) is a frequently diagnosed histologic subtype with increasing morbidity and mortality. RalGDS-Like 4 (RGL4) has not been reported to be associated with cancer risk, prognosis, immunotherapy or any other treatments. We perform a bioinformatics analysis on data downloaded from the Cancer Genome Atlas (TCGA)-LUAD, and we find that low expression of RGL4 is accompanied by worse outcomes and prognosis in LUAD patients. As a promising predictor, the potential influence and mechanisms of RGL4 on overall survival are worth exploring. Moreover, RGL4 expression is significantly associated with a variety of tumor-infiltrating immune cells (TIICs), particularly memory B cells, CD8+T cells and neutrophils. Subsequently, we evaluated the most notable KEGG pathways, including glycolysis gluconeogenesis, the P53 signaling pathway, RNA degradation, and the B cell receptor signaling pathway, among others. Our findings provide evidence that the decreased expression of RGL4 is significantly associated with poor prognosis and immune cell infiltration in patients with LUAD and highlight the use of RGL4 as a novel predictive biomarker for the prognosis of LUAD and other cancers. RGL4 may also be used in combination with immune checkpoints to identify the benefits of immunotherapy. Subjects: Bioinformatics, Genomics, Oncology, Thoracic surgery.


Subject(s)
Adenocarcinoma/metabolism , B-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Lung Neoplasms/metabolism , Lung/immunology , ral Guanine Nucleotide Exchange Factor/metabolism , Adenocarcinoma/diagnosis , Adenocarcinoma/mortality , Cell Movement , Female , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/mortality , Male , Middle Aged , Prognosis , Signal Transduction , Survival Analysis , Tumor Suppressor Protein p53/metabolism , ral Guanine Nucleotide Exchange Factor/genetics
2.
Exp Eye Res ; 185: 107688, 2019 08.
Article in English | MEDLINE | ID: mdl-31185219

ABSTRACT

Photoreceptor degeneration is a significant cause of visual impairment in the United States and globally. Cell replacement therapy shows great promise in restoring vision by transplanting stem-like cells into the sub-retinal space as substitutes for damaged photoreceptors. However, vision repair via transplantation has been limited, in large part, by low numbers of replacement cells able to migrate into damaged retinal tissue and integrate with native photoreceptors. Projects have used external chemical fields and applied electric fields to induce the chemotaxis and electrotaxis of replacement cells, respectively, with limited success. However, the application of combined electro-chemotactic fields in directing cells within biomaterials and host tissue has been surprisingly understudied. The current work examined the ability of combined electro-chemotactic fields to direct the migration of transplantable retinal progenitor cells (RPCs) in controlled microenvironments. Experiments used our established galvano-microfluidic system (Gal-MµS) to generate tunable chemotactic concentration fields with and without superimposed electric fields. Result illustrate that combination fields increased the distance migrated by RPCs by over three times that seen in either field, individually, and with greater directionality towards increasing gradients. Interestingly, immunofluorescence assays showed no significant differences in the distribution of the total and/or activated cognate receptor of interest, indicating that changes in ligand binding alone were not responsible for the measured increases in migration. Bioinformatics analysis was then performed to identity potential, synergistic mechanistic pathways involved in the electro-chemotaxis measured. Results indicate that increased RPC migration in electro-chemotactic fields may arise from down-regulation of cell adhesion proteins in tandem with up-regulation of cytoskeletal regulation proteins. These comprehensive results point towards a novel migration-targeted treatment that may dramatically improve transplantation outcomes as well as elucidate unreported synergy across biological mechanisms in response to electro-chemotactic fields.


Subject(s)
Cell Movement/physiology , Chemotaxis/physiology , Electromagnetic Fields , Retina/cytology , Stem Cell Transplantation , Stem Cells/cytology , Animals , Cadherins/metabolism , Cells, Cultured , DNA Topoisomerases, Type II/genetics , Gene Expression , Immunohistochemistry , Lab-On-A-Chip Devices , Mice , Mice, Inbred C57BL , Poly-ADP-Ribose Binding Proteins/genetics , Real-Time Polymerase Chain Reaction , Receptors, CXCR4/genetics , Stem Cells/physiology , beta Catenin/metabolism , ral Guanine Nucleotide Exchange Factor/genetics
3.
PLoS One ; 11(12): e0167145, 2016.
Article in English | MEDLINE | ID: mdl-27936046

ABSTRACT

RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular ß-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.


Subject(s)
Carrier Proteins/metabolism , GTP Phosphohydrolases/metabolism , Membrane Proteins/metabolism , Monomeric GTP-Binding Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Apoptosis Regulatory Proteins , Binding Sites/genetics , Binding, Competitive , Carrier Proteins/chemistry , Carrier Proteins/genetics , Class I Phosphatidylinositol 3-Kinases , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , Humans , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Models, Molecular , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/genetics , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Domains , Protein Structure, Secondary , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Sequence Homology, Amino Acid , Signal Transduction , ral Guanine Nucleotide Exchange Factor/chemistry , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
4.
Immunity ; 45(1): 94-105, 2016 07 19.
Article in English | MEDLINE | ID: mdl-27438768

ABSTRACT

Although the intracellular trafficking system is integral to most physiologic activities, its role in mediating immune responses to infection has remained elusive. Here, we report that infected bladder epithelial cells (BECs) mobilized the exocyst complex, a powerful exporter of subcellular vesicles, to rapidly expel intracellular bacteria back for clearance. Toll-like receptor (TLR) 4 signals emanating from bacteria-containing vesicles (BCVs) were found to trigger K33-linked polyubiquitination of TRAF3 at Lys168, which was then detected by RalGDS, a guanine nucleotide exchange factor (GEF) that precipitated the assembly of the exocyst complex. Although this distinct modification of TRAF3 served to connect innate immune signaling to the cellular trafficking apparatus, it crucially ensured temporal and spatial accuracy in determining which among the many subcellular vesicles was recognized and selected for expulsion in response to innate immune signaling.


Subject(s)
Escherichia coli/immunology , Immunity, Innate , TNF Receptor-Associated Factor 3/metabolism , Transport Vesicles/metabolism , Urinary Bladder/pathology , Urinary Tract Infections/immunology , Urothelium/immunology , Animals , Cells, Cultured , Escherichia coli/genetics , Exocytosis , Female , Humans , Intracellular Space , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , RNA, Small Interfering/genetics , Signal Transduction , TNF Receptor-Associated Factor 3/genetics , Toll-Like Receptor 4/genetics , Ubiquitination , Urinary Bladder/microbiology , Urothelium/microbiology , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
5.
J Cell Sci ; 128(16): 2957-64, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-26240175

ABSTRACT

The exocyst is an octameric protein complex that is implicated in the tethering of secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. Spatial and temporal control of exocytosis through the exocyst has a crucial role in a number of physiological processes, such as morphogenesis, cell cycle progression, primary ciliogenesis, cell migration and tumor invasion. In this Cell Science at a Glance poster article, we summarize recent works on the molecular organization, function and regulation of the exocyst complex, as they provide rationales to the involvement of this complex in such a diverse array of cellular processes.


Subject(s)
Cell Membrane/metabolism , Exocytosis/genetics , Membrane Fusion Proteins/genetics , Membrane Fusion/genetics , Animals , Cell Cycle/genetics , Cell Membrane/genetics , Humans , Membrane Fusion Proteins/metabolism , Morphogenesis/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
6.
Proc Natl Acad Sci U S A ; 112(21): 6625-30, 2015 May 26.
Article in English | MEDLINE | ID: mdl-25941399

ABSTRACT

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.


Subject(s)
Genes, ras , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Amino Acid Sequence , Binding Sites/genetics , Humans , Lipid Bilayers , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins p21(ras)/chemistry , Sequence Homology, Amino Acid , Signal Transduction , Static Electricity , ral Guanine Nucleotide Exchange Factor/chemistry , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
7.
Asian Pac J Cancer Prev ; 14(5): 2699-701, 2013.
Article in English | MEDLINE | ID: mdl-23803017

ABSTRACT

The RAS family genes encode small GTP-binding cytoplasmic proteins. Activated KRAS engages multiple effector pathways, notably the RAF-mitogen-activated protein kinase, phosphoinositide-3-kinase (PI3K) and RalGDS pathways. In the clinical field, K-ras oncogene activation is frequently found in human cancers and thus may serve as a potential diagnostic marker for cancer cells in circulation. This mini-review aims to summarise information on Ras-induced oncogenesis and the clinical significance of K-ras.


Subject(s)
Adenocarcinoma/pathology , Carcinogenesis/pathology , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Adenocarcinoma/genetics , Humans , Mutation , Pancreatic Neoplasms/genetics , Phosphatidylinositol 3-Kinases/genetics , raf Kinases/genetics , ral Guanine Nucleotide Exchange Factor/genetics
8.
J Mol Cell Cardiol ; 59: 128-38, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23473774

ABSTRACT

Recent work has demonstrated that autophagy, a phylogenetically conserved, lysosome-mediated pathway of protein degradation, is a key participant in pathological cardiac remodeling. One common feature of cell growth and autophagy is membrane biogenesis and processing. The exocyst, an octomeric protein complex involved in vesicle trafficking, is implicated in numerous cellular processes, yet its role in cardiomyocyte plasticity is unknown. Here, we set out to explore the role of small G protein-dependent control of exocyst function and membrane trafficking in stress-induced cardiomyocyte remodeling and autophagy. First, we tested in cultured neonatal rat cardiomyocytes (NRCMs) two isoforms of Ral (RalA, RalB) whose actions are mediated by the exocyst. In these experiments, mTOR inhibition in response to starvation or Torin1 was preserved despite RalA or RalB knockdown; however, activation of autophagy was suppressed only in NRCMs depleted of RalB, implicating RalB as being required for mTOR-dependent cardiomyocyte autophagy. To define further the role of RalB in cardiomyocyte autophagy, we analyzed hearts from mice lacking RalGDS (Ralgds(-/-)), a guanine exchange factor (GEF) for the Ral family of small GTPases. RalGDS-null hearts were similar to wild-type (WT) littermates in terms of ventricular structure, contractile performance, and gene expression. However, Ralgds(-/-) hearts manifested a blunted growth response (p<0.05) to TAC-mediated pressure-overload stress. Ventricular chamber size and contractile performance were preserved in response to TAC in Ralgds(-/-) mice, and load-induced cardiomyocyte autophagy was suppressed. Interestingly, TAC-induced activation of the fetal gene program was similar in both genotypes despite the relative lack of hypertrophic growth in mutant hearts. Together, these data implicate RalGDS-mediated induction of autophagy and exocyst function as a critical feature of load-induced cardiac hypertrophy.


Subject(s)
Autophagy/physiology , Cardiomegaly/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , Animals , Autophagy/genetics , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/genetics
9.
Lung Cancer ; 79(3): 228-35, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23276504

ABSTRACT

BACKGROUND: Thymic epithelial tumors include several entities with different biologic behavior. Chemotherapy is indicated in advanced disease, but limited data exist on gene expression correlation with the response to chemotherapeutic agents. PATIENTS AND METHODS: A series of 69 thymic neoplasms (7 A-, 6 AB-, 6 B1-, 10 B2-, 14 B3-thymomas, 22 carcinomas and 4 combined tumors) was collected to assess gene expression of thymidylate synthase (TS), excision repair cross complementing-1 (ERCC1), ribonucleotide reductase subunit 1 (RRM1), topoisomerase 2α (TOP2A) and mTOR. RESULTS: A strong linear correlation between TS gene and protein expression was observed (P<0.0001, R=0.40). TS expression was significantly lower in pure A-thymomas and thymic carcinomas (P<0.0001) and progressively decreasing from B1-type to thymic carcinomas (B1>B2>B3>C; P<0.0001). RRM1 and TOP2A mRNA expression levels were significantly correlated with TS levels (both P=0.03) with a similar trend of expression among histotypes. RRM1 and TOP2A high levels were significantly correlated with high TS (P=0.03) and low tumor stages (I-II) (P<0.0001 and P<0.01, respectively). No relevant changes of ERCC1 and mTOR were detected. CONCLUSIONS: Low TS and, to a minor extent, RRM1 and TOP2A expression were detected in aggressive thymic tumors. These findings should be prospectively considered in selecting the most appropriate chemotherapy.


Subject(s)
DNA Repair/genetics , Thymidylate Synthase/metabolism , Thymoma/pathology , Thymus Neoplasms/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Cell Transformation, Neoplastic , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Poly-ADP-Ribose Binding Proteins , RNA, Messenger/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Thymidylate Synthase/genetics , Thymoma/drug therapy , Thymoma/enzymology , Thymoma/genetics , Thymus Neoplasms/drug therapy , Thymus Neoplasms/enzymology , Thymus Neoplasms/genetics , Young Adult , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
10.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 34(4): 313-8, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22954110

ABSTRACT

OBJECTIVE: To construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown. METHODS: We chose the binding domain of Raf-1, PI3K, RalGDS, and the function domain of F-Box as well as the U-Box to construct the plasmids. Then used the double enzyme, PCR, and sequence to test the validity and integrity of the cloned nucleotide fragments. The expression efficiency of the plasmids in eukaryotic cells was detected by Western blot analysis. RESULTS: Five of 6 plasmids in this study expressed the corresponding fusion proteins in HEK293T cells, and (RBD+CRD)(Raf-1)- U-Box-pcDNA3.1 can knocked down the protein level of Ras in PANC-1 cells. CONCLUSIONS: We successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.


Subject(s)
Genetic Vectors , Recombinant Fusion Proteins/genetics , Ubiquitin-Protein Ligases/genetics , ras Proteins/genetics , Cloning, Molecular , HEK293 Cells , Humans , Phosphatidylinositol 3-Kinases/genetics , Plasmids , Recombinant Fusion Proteins/metabolism , Transfection , ral Guanine Nucleotide Exchange Factor/genetics
11.
Protein Cell ; 2(4): 308-19, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21494904

ABSTRACT

The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.


Subject(s)
Recombinant Proteins , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor , Amino Acid Sequence , Binding Sites , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , ral GTP-Binding Proteins/chemistry , ral GTP-Binding Proteins/genetics , ral Guanine Nucleotide Exchange Factor/chemistry , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
12.
Oncogene ; 30(34): 3661-71, 2011 Aug 25.
Article in English | MEDLINE | ID: mdl-21441953

ABSTRACT

The Ras superfamily of GTPases is involved in the modification of many cellular processes including cellular motility, proliferation and differentiation. Our laboratory has previously identified the RalGDS-related (Rgr) oncogene in a DMBA (7,12-dimethylbenz[α]anthracene)-induced rabbit squamous cell carcinoma and its human orthologue, hRgr. In this study, we analyzed the expression levels of the human hRgr transcript in a panel of human hematopoietic malignancies and found that a truncated form (diseased-truncated (Dtr-hrgr)) was significantly overexpressed in many T-cell-derived neoplasms. Although the Rgr proto-oncogene belongs to the RalGDS family of guanine nucleotide exchange factors (GEFs), we show that upon the introduction of hRgr into fibroblast cell lines, it is able to elicit the activation of both Ral and Ras GTPases. Moreover, in vitro guanine nucleotide exchange assays confirm that hRgr promotes Ral and Ras activation through GDP dissociation, which is a critical characteristic of GEF proteins. hRgr has guanine nucleotide exchange activity for both small GTPases and this activity was reduced when a point mutation within the catalytic domain (CDC25) of the protein, (cd) Dtr-hRgr, was utilized. These observations prompted the analysis of the biological effects of hRgr and (cd) hRgr expression in cultured cells. Here, we show that hRgr increases proliferation in low serum, increases invasion, reduces anchorage dependence and promotes the progression into the S phase of the cell cycle; properties that are abolished or severely reduced in the presence of the catalytic dead mutant. We conclude that the ability of hRgr to activate both Ral and Ras is responsible for its transformation-inducing phenotype and it could be an important contributor in the development of some T-cell malignancies.


Subject(s)
Cell Transformation, Neoplastic , Leukemia, T-Cell/genetics , Oncogenes , ral Guanine Nucleotide Exchange Factor/genetics , Humans , Immunophenotyping , Proto-Oncogene Mas , RNA, Messenger/genetics
13.
J Phys Chem B ; 114(46): 15331-44, 2010 Nov 25.
Article in English | MEDLINE | ID: mdl-20964430

ABSTRACT

Electrostatic fields at the interface of the Ras binding domain of the protein Ral guanine nucleotide dissociation stimulator (RalGDS) with the structurally analogous GTPases Ras and Rap1A were measured with vibrational Stark effect (VSE) spectroscopy. Eleven residues on the surface of RalGDS that participate in this protein-protein interaction were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe in the form of the thiocyanate (SCN) functional group. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations and solutions to the Poisson-Boltzmann equation using Boltzmann-weighted structural snapshots from molecular dynamics simulations. We found a weak negative correlation between SASA and measured absorption energy, indicating that water exposure of protein surface amino acids can be estimated from experimental measurement of the magnitude of the thiocyanate absorption energy. We found no correlation between calculated field and measured absorption energy. These results highlight the complex structural and electrostatic nature of the protein-water interface. The SCN-labeled RalGDS was incubated with either wild-type Ras or wild-type Rap1A, and the formation of the docked complex was confirmed by measurement of the dissociation constant of the interaction. The change in absorption energy of the thiocyanate functional group due to complex formation was related to the change in electrostatic field experienced by the nitrile functional group when the protein-protein interface forms. At some locations, the nitrile experiences the same shift in field when bound to Ras and Rap1A, but at others, the change in field is dramatically different. These differences identify residues on the surface of RalGDS that direct the specificity of RalGDS binding to its in vivo binding partner, Rap1A, through an electrostatic mechanism.


Subject(s)
Spectrum Analysis/methods , ral Guanine Nucleotide Exchange Factor/chemistry , rap1 GTP-Binding Proteins/chemistry , ras Proteins/chemistry , Binding Sites , Models, Molecular , Molecular Dynamics Simulation , Molecular Probes/chemistry , Molecular Probes/metabolism , Molecular Structure , Protein Binding , Protein Conformation , Static Electricity , Thiocyanates/chemistry , Vibration , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolism , ras Proteins/genetics , ras Proteins/metabolism
14.
Cell Biol Int ; 34(5): 513-22, 2010 Apr 01.
Article in English | MEDLINE | ID: mdl-20067447

ABSTRACT

BRAK/CXCL14 (breast- and kidney-expressed chemokine/CXC chemokine ligand 14) is a chemokine that is expressed in many normal cells and tissues but is absent from or expressed at very low levels in transformed cells and cancerous tissues, including HNSCC (head and neck squamous cell carcinoma). We reported previously that the forced expression of BRAK/CXCL14 in HNSCC (HSC-3 BRAK) cells decreased the rate of tumour formation and size of tumour xenografts compared with mock-vector-introduced (HSC-3 Mock) cells in athymic nude mice, even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that high-level expression of the gene is important for the suppression of tumour establishment in vivo. For the first step to study the mechanisms of BRAK-dependent tumour suppression, we compared characteristics between HSC-3 BRAK and HSC-3 Mock cells under in vitro culture conditions. The cell migration rate was lower in HSC-3 BRAK cells than in HSC-3 Mock cells. Also, HSC-3 BRAK cells showed more rapid adhesion than HSC-3 Mock cells when cultured on type I collagen-coated dishes but not on fibronectin or laminin 1-coated ones. This adhesion was mediated by alpha2beta1 integrin. Immunofluorescent analysis of the cells cultured on type I collagen showed that HSC-3 BRAK cells formed much more elongated focal adhesions co-localized with paxillin and actin stress fibres than did HSC-3 Mock cells. Treatment of parental HSC-3 cells with recombinant BRAK stimulated the activation of Rap1, which is a ras family small GTPase, and formation of elongated focal adhesions, indicating that the difference in cell character observed between HSC-3 Mock and HSC-3 BRAK was not due to selection of clones of different character but due to expression of BRAK in the cells. The characteristic morphology of focal adhesions in HSC-3 BRAK cells was perturbed by the introduction of an expression vector of the Rap-binding domain of the Ral guanine nucleotide dissociation stimulator, a target of Rap1, into HSC-3 BRAK cells, suggesting that Rap1 regulated the formation of the morphology of the focal adhesions. These data indicate that the expression of BRAK stimulated the formation of elongated focal adhesions of the HSC-3 cells in an autocrine or paracrine fashion, in which stimulation may be responsible for the reduced migration of the cells.


Subject(s)
Cell Adhesion/physiology , Cell Movement/physiology , Chemokines, CXC/metabolism , Collagen Type I/metabolism , Focal Adhesions , Tongue Neoplasms , Animals , Apoptosis Regulatory Proteins , Cell Communication/physiology , Cell Line, Tumor , Chemokines, CXC/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/metabolism , Focal Adhesions/ultrastructure , GTP-Binding Proteins , Humans , Integrin alpha2beta1/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Nude , Paxillin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tongue Neoplasms/metabolism , Tongue Neoplasms/pathology , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism , rac1 GTP-Binding Protein/metabolism , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism , rap1 GTP-Binding Proteins/metabolism
15.
Blood ; 112(1): 56-63, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18417737

ABSTRACT

The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.


Subject(s)
Exocytosis/physiology , Weibel-Palade Bodies/physiology , ral Guanine Nucleotide Exchange Factor/physiology , Amino Acid Sequence , Binding Sites/genetics , Calmodulin/metabolism , Cell Line , Endothelial Cells/drug effects , Endothelial Cells/physiology , Endothelial Cells/ultrastructure , Epinephrine/pharmacology , Exocytosis/drug effects , Genetic Variation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , RNA Interference , RNA, Small Interfering/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Thrombin/pharmacology , Transfection , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/antagonists & inhibitors , ral Guanine Nucleotide Exchange Factor/chemistry , ral Guanine Nucleotide Exchange Factor/genetics
16.
Mol Cell Biol ; 28(9): 2851-9, 2008 May.
Article in English | MEDLINE | ID: mdl-18285454

ABSTRACT

The Akt kinase is a key regulator of cell proliferation and survival. It is activated in part by PDK1-induced phosphorylation. Here we show that RalGDS, a Ras effector protein that activates Ral GTPases, has a second function that promotes Akt phosphorylation by PDK1 by bringing these two kinases together. In support of this conclusion is our finding that suppression of RalGDS expression in cells inhibits both epidermal growth factor and insulin-induced phosphorylation of Akt. Moreover, while PDK1 complexes with N-GDS, Akt complexes with the central region of RalGDS through an intermediary, JIP1. The biological significance of this newly discovered RalGDS function is highlighted by the observation that an N-terminally deleted mutant of RalGDS that retains the ability to activate Ral proteins but loses the ability to activate Akt also fails to promote cell proliferation. Thus, RalGDS forms a nexus that transduces growth factor signaling to both Ral GTPase and Akt-mediated signaling cascades.


Subject(s)
Proto-Oncogene Proteins c-akt/metabolism , ral Guanine Nucleotide Exchange Factor/physiology , Animals , Cell Line , Cell Proliferation , Enzyme Activation , Epidermal Growth Factor/metabolism , Humans , Insulin/metabolism , Mice , Mutation , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Signal Transduction , ral Guanine Nucleotide Exchange Factor/genetics
17.
Neuron ; 56(4): 604-20, 2007 Nov 21.
Article in English | MEDLINE | ID: mdl-18031680

ABSTRACT

To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.


Subject(s)
Growth Cones/metabolism , Microtubules/metabolism , Motor Neurons/metabolism , Spinal Cord/abnormalities , Spinal Cord/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , Animals , Cell Differentiation/genetics , Cells, Cultured , Efferent Pathways/abnormalities , Efferent Pathways/cytology , Efferent Pathways/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Developmental/genetics , Genetic Testing , Growth Cones/ultrastructure , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Microtubules/ultrastructure , Motor Neurons/cytology , Mutation/genetics , Paclitaxel/pharmacology , Spinal Cord/cytology , Tubulin Modulators/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism , ral Guanine Nucleotide Exchange Factor/genetics
19.
Cell Signal ; 19(7): 1575-82, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17382517

ABSTRACT

A cDNA encoding a RalGDS-related protein, Rgl3, was isolated by yeast two-hybrid screening using a small G-protein, Rap1, as a bait. Rgl3 mRNA is commonly detectable in several visceral organs (e.g. kidney, heart, liver, and lung) in the mouse and human. The Rgl3 protein mainly localizes in the cytoplasm when expressed in fibroblasts. Yeast two-hybrid assay indicated that Rgl3 could interact with Rap1, Rap2, H-Ras, N-Ras, and R-Ras but failed to interact efficiently with Ral and Rho. Interestingly, Rgl3 was found to affect cell morphology in two assay systems in culture. First, Rgl3 suppressed cell-spreading induced by Rap1, R-Ras, or C3G-CAAX (a membrane-targeted Rap/R-Ras activator) in HEK-293 cells. Second, Rgl3 enhanced the focus-formation induced by oncogenic H-Ras and N-Ras mutants in NIH3T3 cells. Moreover, we identified profilin II as a potential binding partner for Rgl3 by yeast two-hybrid screening. This interaction requires the characteristic proline cluster in the Rgl3 amino-terminal domain. Profilin II and Rgl3 co-operated in enhancing the N-Ras-induced focus-formation. These findings raise the possibility that Rgl3 mediates interaction between Ras/Rap-family proteins and profilin II, an important activator of actin polymerization.


Subject(s)
Profilins/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , rap1 GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Cell Line, Transformed , Cell Movement , Gene Expression Profiling , Gene Expression Regulation , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Subcellular Fractions/metabolism , ral Guanine Nucleotide Exchange Factor/chemistry , ral Guanine Nucleotide Exchange Factor/genetics , ras Proteins/metabolism
20.
Mol Cell Biol ; 26(10): 3966-75, 2006 May.
Article in English | MEDLINE | ID: mdl-16648489

ABSTRACT

Hyperactivation of Ras is one of the most common abnormalities in acute myeloid leukemia. In experimental models, Ras inhibits myeloid differentiation, which is characteristic of leukemia; however, the mechanism through which it disrupts hematopoiesis is poorly understood. In multipotent FDCP-mix cells, Ras inhibits terminal neutrophil differentiation, thereby indefinitely extending their proliferative potential. Ras also strongly promotes the sensitivity of these cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). Using this model, we have dissected the signaling elements downstream of Ras to determine their relative contribution to the dysregulation of hematopoiesis. Cells expressing Ras mutants selectively activating Raf (Ras*T35S) or phosphatidylinositol 3-kinase (Ras*Y40C) did not significantly affect differentiation or proliferative capacity, whereas Ras*E37G (which selectively activates RalGEFs) perpetuated proliferation and blocked neutrophil development in a manner similar to that of Ras. Correspondingly, expression of constitutively active versions of these effectors confirmed the overriding importance of Ral guanine nucleotide exchange factors. Cells expressing Ras demonstrated hyperactivation of Ral, which itself was able to exactly mimic the phenotype of Ras, including hypersensitivity to GM-CSF. Conversely, dominant negative Ral promoted spontaneous neutrophil development. Ral, in turn, appears to influence differentiation through multiple effectors. These data show, for the first time, the importance of Ral in regulating differentiation and self-renewal in hematopoietic cells.


Subject(s)
Cell Differentiation , Genes, ras , Leukemia, Myeloid/physiopathology , ral Guanine Nucleotide Exchange Factor/physiology , ras Proteins/physiology , Animals , Blotting, Western , Cell Culture Techniques , Cell Line, Tumor , Cell Proliferation , Clone Cells , Dose-Response Relationship, Drug , Enzyme Activation , Gene Expression Regulation, Leukemic , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Leukemia, Myeloid/genetics , Mice , Mutation , Neutrophils/cytology , Neutrophils/metabolism , Retroviridae/genetics , ral Guanine Nucleotide Exchange Factor/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...