Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 89
Filter
Add more filters










Publication year range
1.
Mol Cell Biochem ; 479(3): 567-577, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37131040

ABSTRACT

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.


Subject(s)
Quinine , ral Guanine Nucleotide Exchange Factor , ral Guanine Nucleotide Exchange Factor/metabolism , Quinine/pharmacology , Guanine Nucleotide Exchange Factors/metabolism , Epithelial Cells/metabolism
2.
Acta Histochem ; 124(7): 151938, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35981451

ABSTRACT

RILP (Rab-interacting lysosomal protein) is a key regulator of lysosomal transport and a potential tumor suppressor. However, the role of RILP in prostate cancer and the underlying mechanism of RILP in regulating the proliferation, migration, and invasion of prostate cancer cells remain to be studied. In this study, we confirmed RalGDS (Ral guanine nucleotide dissociation stimulator) as the interaction partner of RILP in PC3 prostate cancer cells. Immunofluorescence microscopy showed that RILP recruits RalGDS to the lysosomal compartment. We found that RILP inhibits the activation of RalA and downstream effector RalBP1, and negatively regulates the downstream molecular phosphorylation of Ras. We showed that RILP inhibits the proliferation, migration, and invasion of PC3 prostate cancer cells, which may give rise to novel ideas for cancer treatment.


Subject(s)
Prostatic Neoplasms , ral Guanine Nucleotide Exchange Factor , Adaptor Proteins, Signal Transducing/metabolism , Cell Proliferation , Guanine Nucleotides , Humans , Male , PC-3 Cells , ral Guanine Nucleotide Exchange Factor/metabolism
3.
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
4.
J Exp Clin Cancer Res ; 38(1): 391, 2019 Sep 05.
Article in English | MEDLINE | ID: mdl-31488180

ABSTRACT

BACKGROUND: Clinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. Myc-associated zinc-finger protein (MAZ) is a well-documented oncogene involved in the progression and metastasis of multiple cancer types, even in PCa. However, the clinical significance and biological roles of MAZ in bone metastasis of PCa remain unclear. METHODS: MAZ expression was examined in PCa tissues with bone metastasis, PCa tissues without bone metastasis and metastatic bone tissues by real-time PCR and immunohistochemistry (IHC), respectively. Statistical analysis was performed to evaluate the clinical correlation between MAZ expression and clinicopathological features and bone metastasis-free survival in PCa patients. Biological roles of MAZ in bone metastasis of PCa were investigated both in vitro by transwell assay, and in vivo by a mouse model of left cardiac ventricle inoculation. The bioinformatics analysis, western blot, pull-down assays, chromatin immunoprecipitation (ChIP) and luciferase reporter assays were applied to demonstrate and examine the relationship between MAZ and its potential downstream signalling pathway. TaqMan copy number assay was performed to identify the underlying mechanism responsible for MAZ overexpression in PCa tissues. RESULTS: MAZ expression is elevated in PCa tissues with bone metastasis compared with that in PCa tissues without bone metastasis, and is further increased in metastatic bone tissues. High expression of MAZ positively correlates with poor overall and bone metastasis-free survival in PCa patients. Upregulating MAZ elevates, while silencing MAZ represses the invasion and migration abilities of PCa cells in vitro and bone metastasis ability in vivo. Our results further reveal that MAZ promotes bone metastasis of PCa dependent on KRas signalling, although MAZ transcriptionally upregulates KRas and HRas expression, where the Ral guanine nucleotide exchange factor (RalGEF) signaling is responsible for the different roles of KRas and HRas in mediating the pro-bone metastasis of MAZ in PCa. Finally, our results indicate that recurrent gains contribute to MAZ overexpression in a small portion of PCa tissues. CONCLUSION: These results indicate that the MAZ/Kras/ RalGEF signalling axis plays a crucial role in promoting PCa cell bone metastasis, suggesting a potential therapeutic utility of MAZ in bone metastasis of PCa.


Subject(s)
Bone Neoplasms/secondary , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Transcription Factors/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , Aged , Animals , Biopsy , Bone Neoplasms/mortality , Cell Line, Tumor , Disease Models, Animal , Humans , Immunohistochemistry , Male , Mice , Models, Biological , Neoplasm Grading , Neoplasm Staging , Signal Transduction
5.
Methods Mol Biol ; 1957: 169-175, 2019.
Article in English | MEDLINE | ID: mdl-30919354

ABSTRACT

G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which in addition to signaling via heterotrimeric G proteins can activate small G proteins both directly and indirectly. The activation of a variety of GPCRs leads to the translocation of Ral GDP dissociation stimulator (RalGDS) to the plasma membrane, where it functions as a guanine nucleotide exchange factor of RalA to promote membrane blebbing. The translocation of RalGDS is ß-arrestin-dependent and can be inhibited by either the expression of the ß-arrestin1 amino-terminal domain or the expression of RalGDS clone 284 (amino acid residues 616-768 of RalGDS). We describe here a methodology for assessing GPCR-dependent stimulation of RalGDS plasma membrane translocation.


Subject(s)
Cell Membrane/metabolism , Molecular Biology/methods , Receptors, G-Protein-Coupled/metabolism , beta-Arrestins/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , Data Analysis , HEK293 Cells , Humans , Receptor, Angiotensin, Type 1/metabolism
6.
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
7.
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
8.
Clin Cancer Res ; 22(1): 230-242, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26283684

ABSTRACT

PURPOSE: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear. EXPERIMENTAL DESIGN: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models. RESULTS: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells. CONCLUSIONS: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target.


Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Gene Expression , Receptor, EphA2/genetics , Biomarkers, Tumor , Cell Line, Tumor , Cell Movement/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Humans , Kaplan-Meier Estimate , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Proportional Hazards Models , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Receptor, EphA2/metabolism , Reproducibility of Results , Signal Transduction , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , ras Proteins/metabolism
9.
Cell Death Dis ; 6: e1923, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26469971

ABSTRACT

RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Breast Neoplasms/metabolism , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , Breast Neoplasms/pathology , Cell Movement , Cell Proliferation , Endosomes/metabolism , Female , Humans , MAP Kinase Signaling System , MCF-7 Cells , Neoplasm Invasiveness , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , ral Guanine Nucleotide Exchange Factor/chemistry
10.
Expert Rev Proteomics ; 12(6): 669-82, 2015.
Article in English | MEDLINE | ID: mdl-26496174

ABSTRACT

Illustrated here is the critical role of oncogenic KRAS in the initiation of cancer through deregulation of the G1 cell cycle, and elements and scenarios taking place under physiological conditions and in KRAS-driven cancer. Raf, PI3K and RalGDS are major K-Ras effectors. They bind at the same Ras site. What decides the cell selection among them? This temporal and spatial decision is critical since in some cellular context the outcome of their signaling pathways may oppose each other. Key among them is the concentration of calcium/calmodulin, negative feedback loops, where a downstream member of the pathway inhibits its upstream activator and cross-inhibition, where inhibition entails blocking another pathway. These three elements, in addition to spatial restrictions by K-Ras-membrane interactions, are not independent; they integrate to provide blueprints for cell decisions. Importantly, elucidation of signaling requires not only K-Ras binary interactions; but the structures and dynamics of its multiprotein complexes.


Subject(s)
Carcinogenesis/metabolism , G1 Phase Cell Cycle Checkpoints , Proto-Oncogene Proteins p21(ras)/metabolism , Animals , Humans , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , raf Kinases/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism
11.
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
12.
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
13.
Carcinogenesis ; 36(1): 2-12, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25239643

ABSTRACT

Cancer is a multistep disease that begins with malignant cell transformation and frequently culminates in metastasis. MicroRNAs (miRNAs) are small regulatory 21-25 nt RNA molecules and are frequently deregulated in cancer. miR-200a is a member of the miR-200 family, which are known inhibitors of the epithelial-to-mesenchymal transition. As such, the tumor-suppressive role of miR-200a in oncogenesis has been well documented; however, recent studies have found a proliferative role for this miRNA as well as a prometastatic role in the later steps of cancer progression. Little is known about the role of this miRNA in the early stages of cancer, namely, malignant cell transformation. Here, we show that miR-200a alone transforms an immortalized rat epithelial cell line, and miR-200a cooperates with Ras to enhance malignant transformation of an immortalized human epithelial cell line. Furthermore, miR-200a induces cell transformation and tumorigenesis in immunocompromised mice by cooperating with a Ras mutant that activates only the RalGEF effector pathway, but not Ras mutants activating PI3K or Raf effector pathways. This transformative ability is in accordance with miR-200a targeting Fog2 and p53 to activate Akt and directly repress p53 protein levels, respectively. These results demonstrate an oncogenic role for miR-200a and provide a specific cellular context where miR-200a acts as an oncomiR rather than a tumor suppressor by cooperating with an oncogene in malignant cell transformation.


Subject(s)
Breast Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Epithelial Cells/pathology , Kidney/pathology , MicroRNAs/physiology , Animals , Apoptosis , Blotting, Western , Breast/metabolism , Breast/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Epithelial Cells/metabolism , Female , Humans , Kidney/metabolism , Male , Mice , Mice, Nude , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats , ral Guanine Nucleotide Exchange Factor/metabolism
14.
Phys Chem Chem Phys ; 16(37): 20047-60, 2014 Oct 07.
Article in English | MEDLINE | ID: mdl-25127074

ABSTRACT

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras-Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras-Raf and Ras-RalGDS complexes.


Subject(s)
raf Kinases/chemistry , ral Guanine Nucleotide Exchange Factor/chemistry , ras Proteins/chemistry , Hydrogen Bonding , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Tertiary , Spectrophotometry, Infrared , Static Electricity , raf Kinases/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , ras Proteins/metabolism
15.
Front Med ; 7(4): 452-61, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24264166

ABSTRACT

BCR/ABL is the causative agent of chronic myelogenous leukemia (CML). Through structure/function analysis, several protein motifs have been determined to be important for the development of leukemogenesis. Tyrosine177 of BCR is a Grb2 binding site required for BCR/ABL-induced CML in mice. In the current study, we use a mouse bone marrow transduction/transplantation system to demonstrate that addition of oncogenic NRAS (NRASG12D) to a vector containing a BCR/ABL(Y177F) mutant "rescues" the CML phenotype rapidly and efficiently. To further narrow down the pathways downstream of RAS that are responsible for this rescue effect, we utilize well-characterized RAS effector loop mutants and determine that the RAL pathway is important for rapid induction of CML. Inhibition of this pathway by a dominant negative RAL is capable of delaying disease progression. Results from the present study support the notion of RAL inhibition as a potential therapy for BCR/ABL-induced CML.


Subject(s)
Biomarkers, Tumor/metabolism , Carcinogenesis/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism , ras Proteins/metabolism , Animals , Blotting, Western , Bone Marrow Transplantation , Flow Cytometry , Fluorescent Antibody Technique , Genetic Vectors , Mice , NIH 3T3 Cells , Retroviridae
16.
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
17.
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
18.
Cell Cycle ; 12(3): 522-32, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23324341

ABSTRACT

Mutant K-Ras and survivin both contribute to oncogenesis, but little is known about K-Ras requirement for the maintenance of the high levels of survivin in human tumors. Here we demonstrate that K-Ras depletion significantly decreases survivin levels in human cancer cells that harbor mutant but not wild type K-Ras. K-Ras depletion attenuates both basal and drug-induced survivin levels. The mechanism by which K-Ras depletion decreases survivin levels is through ubiquitination and proteasomal degradation of survivin and is independent of survivin-Thr-34 phosphorylation. Depletion of RalA and RalB, but not Raf-1, Akt1 and Akt2, decreases survivin levels, suggesting that K-Ras may regulate survivin stability through its RalGDS/Ral but not PI3K/Akt and Raf-1/Mek effector pathways. Furthermore, the ability of mutant K-Ras to induce anchorage-independent growth, invasion and survival is compromised by depletion of survivin. These studies suggest that mutant K-Ras contributes to the maintenance of the aberrantly high levels of survivin in tumors by regulating its stability, and that the ability of mutant K-Ras to induce malignant transformation is, at least in part, dependent on these high levels of survivin.


Subject(s)
Inhibitor of Apoptosis Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , 3T3 Cells , Animals , Cell Line , Cell Proliferation , Cell Survival , HEK293 Cells , Humans , Inhibitor of Apoptosis Proteins/genetics , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins p21(ras)/deficiency , RNA Interference , RNA, Small Interfering , Signal Transduction/genetics , Survivin , Ubiquitination , raf Kinases/genetics , raf Kinases/metabolism , ral GTP-Binding Proteins/genetics , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/metabolism
19.
J Phys Chem B ; 116(31): 9326-36, 2012 Aug 09.
Article in English | MEDLINE | ID: mdl-22738401

ABSTRACT

The human protein Rap1A (Rap) is a member of the Ras superfamily of GTPases that binds to the downstream effector Ral guanine nucleotide dissociation stimulator (RalGDS). Although Ras and Rap have nearly identical amino acid sequences and structures along the effector binding surface, the charge reversal mutation Rap K31E has previously been shown to increase the dissociation constant of Rap-RalGDS docking to values similar to that of Ras-RalGDS docking. This indicates that the difference in charge at position 31 could provide a mechanism for Ral to distinguish two structurally similar but functionally distinct GTPases, which would be of vital importance for appropriate biological function. In this report, vibrational Stark effect spectroscopy, dissociation constant measurements, and molecular dynamics simulations were used to investigate the role that electrostatic field differences caused by the charge reversal mutation Rap K31E play in determining the binding specificity of RalGDS to Rap versus Ras. To do this, six variants of RalGDS carrying a thiocyanate electrostatic probe were docked with three Rap mutants, E30D, K31E, and E30D/K31E. The change in absorption energy of the thiocyanate probe caused by RalGDS docking to these Rap variants was then compared to that observed with wild-type Ras. Three trends emerged: the expected reversion behavior, an additive behavior of the two single mutations, and cancelation of the effects of each single mutation in the double mutant. These observations are explained with a physical model of the position of the thiocyanate probe with respect to the mutated residue based on molecular dynamics simulations.


Subject(s)
Point Mutation , ral Guanine Nucleotide Exchange Factor/metabolism , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolism , Humans , Molecular Dynamics Simulation , Protein Binding , Spectrophotometry, Infrared/methods , Static Electricity , Thiocyanates/chemistry , ral Guanine Nucleotide Exchange Factor/chemistry , rap1 GTP-Binding Proteins/chemistry
20.
Mol Cell ; 45(5): 610-8, 2012 Mar 09.
Article in English | MEDLINE | ID: mdl-22326055

ABSTRACT

The connection between cancer and inflammation is widely recognized, yet the underlying molecular mechanisms are poorly understood. We report here that TIPE2 provides a molecular bridge from inflammation to cancer by targeting the Ras signaling pathway. TIPE2 binds the Ras-interacting domain of the RalGDS family of proteins, which are essential effectors of activated Ras. This binding prevented Ras from forming an active complex, thereby inhibiting the activation of the downstream signaling molecules Ral and AKT. Consequently, TIPE2 deficiency led to heightened activation of Ral and AKT, resistance to cell death, increased migration, and dysregulation of exocyst complex formation. Conversely, TIPE2 overexpression induced cell death and significantly inhibited Ras-induced tumorigenesis in mice. Importantly, TIPE2 expression was either completely lost or significantly downregulated in human hepatic cancer. Thus, TIPE2 is an inhibitor of both inflammation and cancer, and a potential drug target for inflammatory and neoplastic diseases.


Subject(s)
Genes, ras , Intracellular Signaling Peptides and Proteins/physiology , Adult , Aged , Aged, 80 and over , Animals , Apoptosis/genetics , Binding Sites , Binding, Competitive , Carcinoma, Hepatocellular/genetics , Cell Movement/genetics , Cell Transformation, Neoplastic/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Liver Neoplasms/genetics , Mice , Mice, Inbred C57BL , Middle Aged , NIH 3T3 Cells , Oncogene Protein v-akt/genetics , ral GTP-Binding Proteins/genetics , ral Guanine Nucleotide Exchange Factor/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...