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ITSN1 and Ruk/CIN85 colocalized to clathrin-coated pits in MCF-7 cells
Nikolaienko O. V.,Skrypkina I. Ya.,Tsyba L. O.,Drobot L. B.
Biopolymers and Cell , 2009,
Abstract: Aim. Activation of receptor tyrosine kinases (RTK) by corresponding ligands results not only in signal propagation, but also initiates a number of processes, such as clathrin-mediated endocytosis, which precisely regulate biological outcome. These processes are tightly controlled by coordinated action of a plethora of proteins – enzymes, scaffolds and inhibitory molecules. An example of an endocytic accessory protein that also functions in cell signaling is provided by intersectin 1 (ITSN1). Previously we have shown that ITSN1 forms a complex with adaptor protein Ruk/CIN85 and ubiquitin ligase Cbl-b, which are implicated in down regulation of RTK. The present study aimed to determine the subcellular localization of ITSN1-Ruk/CIN85 complexes relatively to clathrin light chain and Cbl-b. Methods. Transient transfection of MCF-7 breast adenocarcinoma cells with the constructs containing Omni-tagged intersectin 1 and clathrin light chain fused with mCherry fluorescent protein was utilized to determine subcellular localization by direct or indirect immunofluorescence. Results. We found that Ruk/CIN85-ITSN1 complexes partially colocalized with Cbl-b and clathrin light chain in MCF-7 cells. Conclusions. In our report we provide experimental evidence that ITSN1-Ruk/CIN85 complexes exist in pre-assembled state with Cbl-b and are targeted to clathrin-coated pits in MCF-7 cells
Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus  [PDF]
Dergai M. V.,Dergai O. V.,Tsyba L. O.,Novokhatska O. V.
Biopolymers and Cell , 2011,
Abstract: Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN122a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules.
Endocytic Adaptor Protein Epsin Is Elevated in Prostate Cancer and Required for Cancer Progression  [PDF]
Kandice L. Tessneer,Satish Pasula,Xiaofeng Cai,Yunzhou Dong,Xiaolei Liu,Lili Yu,Scott Hahn,John McManus,Yiyuan Chen,Baojun Chang,Hong Chen
ISRN Oncology , 2013, DOI: 10.1155/2013/420597
Abstract: Epsins have an important role in mediating clathrin-mediated endocytosis of ubiquitinated cell surface receptors. The potential role for epsins in tumorigenesis and cancer metastasis by regulating intracellular signaling pathways has largely not been explored. Epsins are reportedly upregulated in several types of cancer including human skin, lung, and canine mammary cancers. However, whether their expression is elevated in prostate cancer is unknown. In this study, we investigated the potential role of epsins in prostate tumorigenesis using the wild type or epsin-deficient human prostate cancer cells, LNCaP, in a human xenograft model, and the spontaneous TRAMP mouse model in wild type or epsin-deficient background. Here, we reported that the expression of epsins 1 and 2 is upregulated in both human and mouse prostate cancer cells and cancerous tissues. Consistent with upregulation of epsins in prostate tumors, we discovered that depletion of epsins impaired tumor growth in both the human LNCaP xenograft and the TRAMP mouse prostate. Furthermore, epsin depletion significantly prolonged survival in the TRAMP mouse model. In summary, our findings suggest that epsins may act as oncogenic proteins to promote prostate tumorigenesis and that depletion or inhibition of epsins may provide a novel therapeutic target for future prostate cancer therapies. 1. Introduction Solid tumors, such as those in prostate cancer, contribute the majority of all cancers and result in significant distant tumor metastasis to vital organs such as the lungs, brain, and bones [1, 2]. Prostate cancer contributes significantly to the morbidity and mortality of men in the United States [3]. Advanced prostate cancer is associated with significant mortality because the cancer metastasizes and spreads throughout the body, making recovery nearly impossible [1, 4]. The high rates of prostate cancer metastasis are, in part, caused by aggressive primary tumor growth in prostate [5]. Prostate tumorigenesis is a result of several upregulated signaling pathways, including Notch, EGF, FGF, and Wnt signaling, which promote tumor cell proliferation [6–11]. Understanding the mechanisms responsible for upregulated signaling during early tumorigenesis is an important step in identifying key regulators and potential therapeutic targets. More importantly, targeting early stages of tumorigenesis will facilitate stabilization of rapid growing tumors, leading to effective surgical removal of primary tumors and inhibition of further tumor metastasis. Epsins are endocytic adaptor proteins that regulate
The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers  [PDF]
Benedetta Pozzi, Stefania Amodio, Caterina Lucano, Anna Sciullo, Simona Ronzoni, Daniela Castelletti, Thure Adler, Irina Treise, Ingrid Holmberg Betsholtz, Birgit Rathkolb, Dirk H. Busch, Eckhard Wolf, Helmut Fuchs, Valérie Gailus-Durner, Martin Hrabě de Angelis, Christer Betsholtz, Stefano Casola, Pier Paolo Di Fiore, Nina Offenh?user
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0050818
Abstract: Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220+ bone marrow cells, CD19? thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.
Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
Gubar O. S.,Kropyvko S. V.,Tsyba L. O.,Gasman S.
Biopolymers and Cell , 2012,
Abstract: Intersectin 1 (ITSN1) is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S) are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.
Integridade no posicionamento RTK e RTK em rede
Barbosa, Eduardo De Magalh?es;Monico, Jo?o Francisco Galera;Alves, Daniele Barroca Marra;De Oliveira, Leonardo Castro;
Boletim de Ciências Geodésicas , 2010, DOI: 10.1590/S1982-21702010000400007
Abstract: technological advances in positioning methods have enabled the development of methodologies that allow its use in a great variety of applications. one method of gnss (global navigation satellite system) positioning of high performance is the rtk (real time kinematic) whose basic principle takes advantage of the high correlation of the errors caused by ionosphere, troposphere and satellite orbit in a reference station and a nearby station of interest, besides requiring a radio link or other communication system to transmit the data from the base to the user. but as the distances increase, the correlation decreases and the performance is reduced. to reduce this problem, the concept of network stations (rtk network) was developed. such approach uses the infrastructure of a network of reference stations to provide corrections to the network users. the rtk and rtk network allows introducing the concept of integrity, widely used in navigation, in the context of geodesy. in this article, we present the concept of integrity and its use for evaluating the integrity on rtk and rtk network in the context of s?o paulo state gnss network. results showed that to guarantee the integrity displayed by hpl
Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins  [PDF]
Olga Novokhatska, Mykola Dergai, Liudmyla Tsyba, Inessa Skrypkina, Valeriy Filonenko, Jacques Moreau, Alla Rynditch
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070546
Abstract: Background Scaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs. Methodology/Principal Findings We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform. Conclusions/Significance Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.
To RTK points in CZEPOS network  [PDF]
Zdeněk Nevosád
Acta Montanistica Slovaca , 2007,
Abstract: For the purpose of checking RTK points, it is possible to use the repeated measurements, the measurement of quantities between the points, or the quantities within connection surveys. Suitable satellite or terrestrial measurements between RTK and surrounding points of the given network may serve for the homogeneity check. For heights check and more accurate, it is convenient to connect the satellite surveys to the levelling control.
Endocytic Adaptor Protein Epsin Is Elevated in Prostate Cancer and Required for Cancer Progression
Kandice L. Tessneer,Satish Pasula,Xiaofeng Cai,Yunzhou Dong
ISRN Oncology , 2013, DOI: 10.1155/2013/420597
A New Role for Clathrin Adaptor Proteins 1 and 3 in Lipoplex Trafficking  [PDF]
Justine E. Alford, Jade Gumbs, Emma C. Anderson
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0091429
Abstract: Intracellular protein trafficking through secretory and endocytic pathways depends on the function of adaptor proteins that bind motifs on cargo proteins. The adaptor proteins then recruit coat proteins such as clathrin, enabling the formation of a transport vesicle. While studying the role of the clathrin adaptor proteins, AP-1, AP-2 and AP-3 in viral protein trafficking, we discovered that AP-1 and AP-3 potentially have a role in successful transfection of mammalian cells with DNA-liposome complexes (lipoplexes). We showed that AP-1, -2 and -3 are not required for lipoplexes to enter cells, but that lipoplexes and/or released DNA are unable to reach the nucleus in the absence of AP-1 or AP-3, leading to minimal exogenous gene expression. In contrast, gene expression from liposome-delivered mRNA, which does not require nuclear entry, was not impaired by the absence of AP-1 or AP-3. Despite the use of lipoplexes to mediate gene delivery being so widely used in cell biology and, more recently, gene therapy, the mechanism by which lipoplexes or DNA reach the nucleus is poorly characterised. This work sheds light on the components involved in this process, and demonstrates a novel role for AP-1 and AP-3 in trafficking lipoplexes.
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