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Identification and Biochemical Characterization of the Phosphotyrosine- and ShcA-binding Protein STS-1
Get PDFShcA is a protein that is recruited to receptor-protein tyrosine kinases, where it is phosphorylated on several tyrosine residues. These phosphotyrosine residues act as binding sites for Grb2. Interestingly, some receptors also bind Grb2 directly, which would suggest a redundant role for ShcA. Recently, we demonstrated that cells expressing receptors engineered to bind ShcA display enhanced rates of cell division compared to cells expressing versions of the receptor designed to bind Grb2 directly. In this dissertation, I describe our efforts to identify and characterize novel ShcA-binding proteins that may play a role in this phenomenon.Chapter 1 is an introductory chapter that describes the field of receptor protein-tyrosine kinase signaling.Chapter 2 describes the use of ShcA-derived phosphopeptides as reagents to purify proteins that bind ShcA in a phosphorylation-dependent manner. This led to the identification of STS-1 as a protein that associates with the phosphorylated Tyr317 region of ShcA. In cell-based experiments, we demonstrate that the association between STS-1 and ShcA is enhanced following growth factor stimulation.In chapter 3, we demonstrate that STS-1 binds directly to the phosphorylated Tyr317 ShcA-derived peptide. To determine which region of STS-1 directly binds the peptide, two complementary approached were undertaken. In the first, purified STS-1 was subjected to limited proteolysis with thermolysin and the resulting fragments were incubated with the phosphorylated Tyr317 ShcA-derived peptide. A fragment that includes the region of STS-1 with homology to 2H phosphoesterases retained the ability to bind the peptide. In the second approach, mammalian cell lysates expressing STS-1 constructs with point mutations intended to disable individual domains of STS-1 were incubated the phosphorylated Tyr317 ShcA-derived peptide. STS-1 constructs with mutations in the domain with homology to 2HPEs failed to associate with the peptide.Chapter 4 describes the ability of STS-1 to dephosphorylate (or regulate dephosphorylation) of ShcA. We show that STS-1 can dephosphorylate tyrosine residues of ShcA in vitro. Additionally, we show that depletion of STS-1 results in enhanced ShcA tyrosine phosphorylation and enhanced Akt activation in mammalian cells.In summary, my results demonstrate that STS-1 binds directly to ShcA in a phosphotyrosine-dependent manner and that STS-1 may also be a physiologically relevant ShcA-phosphatase
Localization of CSF-1 Receptor intracellular domain in the nucleus after its release from the plasma membrane by regulated intramembrane proteolysis
No full textIncludes bibliographical references (p. 33-38)The Colony Stimulating Factor 1 receptor (CSF-1 receptor) is a protein tyrosine kinase that controls the survival, proliferation, and differentiation of mononuclear phagocytes and regulates cells of the female reproductive tract. Receptor deficient mice show defects in bone formation; development of the mammary gland and lack of macrophages. CSF-1 receptor overexpression has been linked to cancers of the breast, ovaries, and endometrium. We have shown previously that the CSF-1 receptor is subject to regulated intramembrane proteolysis or RIP. RIP involves cleavage at two separate sites in the receptor and results in ectodomain shedding, followed by the release of the intracellular domain (ICD) into the cytosol. We propose that the ICD travels to the nucleus where it is likely to be involved in regulation of proinflammatory gene transcription. To investigate this we have to increase the stability of the ICD by adding an amino-terminal V5-epitope tag. In addition, we have mutated a stretch of leucine and/or conserved lysine residues in the context of the epitopetagged ICD, to investigate their role in localization of the ICD. Normal and mutant proteins were expressed in 293 cells and analyzed for subcellular localization by subcellular fractionation followed by immunoblotting. The normal and mutant protein expressing cell lines were later analyzed by immunofluorescence. The V5-tagged ICD protein has been found in the cytoplasmic fraction and to a lesser extent in nucleus. Mutation of the leucine and conserved lysine residues resulted in an increase in the level of nuclear ICD whereas mutation of the conserved lysine residues resulted in a decrease in the levels of nuclear ICD and also the stability of the overall protein. We used confocal immunofluorescence to confirm the results obtained using subcellular fractionation. While most of the CICD is present in the cytoplasm, the ICD can also be detected in the nucleus. Mutation of the conserved lysines results in a decrease in the levels of the ICD seen in the nucleus and hence may be a part of the export signal. Mutation of a stretch of leucines and conserved lysines results in an increase in the levels of the ICD seen in the nucleus and hence may be a part of the import signal
Structural studies of suppressor of T-cell receptor signaling-1
No full textIncludes bibliographical references (pages 25-26).Multicellular organisms use a multistep process called signal transduction for cell to cell communication. Messaging cells will secrete soluble, extracellular signals to recipient cells. The extracellular signal will bind to cell surface receptors of the recipient cells that then trigger signaling cascades within the cell, to elicit a physiological response. Our interest is in receptor tyrosine kinases (RTKs) and their role in signal transduction. One protein that associates with activated RTKs is Shc. Shc is a receptor binding protein that also interacts with Grb2, a protein at the initial stage of the Ras-MAP Kinase pathway. We have evidence that Shc has more functions than acting as an adaptor for receptors and Grb2. We have identified another Shc binding protein, Suppressor of T-cell receptor Signaling-1 (STS-1) which has not been previously reported. STS-1 bound to Shc in a phosphorylation dependent manner, leading us to look into this protein and investigate how these proteins are binding. There are four domains within STS-1: the amino terminus contains a ubiquitin-associated (UBA) domain, a 2 histidine phosphoesterease (2HPE) domain, and a Src homology 3 (SH3) domain while a phosphoglycerate mutase homology (PGM) domain resides in the carboxyl terminus. There are previous studies indicating that the PGM domain has phosphatase activity and the crystal structure of this domain has been solved. Our focus is determining how STS-1 recognizes phosphotyrosine residues. Based on the Simple Modular Architecture Research Tool (SMART), there are no phosphotyrosine binding domains (SH2, PTB, C2, or HYB) in STS-1. We hypothesize that there must be a novel phosphotyrosine binding domain within STS-1. Using recombinant STS-1 constructs, we plan on using X-ray crystallography to find out which residues in this domain are associating with the tyrosine phosphorylated peptide substrate by comparing unbound and bound crystal structures
A High Throughput Multiplexed Platform for Monitoring Proteolysis in the Classical Secretory Pathway -Search for Novel Antivirals
No full textIncludes bibliographical references (pages 32-35).RNA Viruses such as Human Immunodeficiency (HIV) and Dengue Virus (DenV) cause devastating diseases, including Acquired Immunodeficiency Syndrome (AIDS) and Dengue Hemorrhagic Fever. With 2.5 and 50 million new infections yearly, respectively, unsuccessful attempts at vaccine development and resistance to current treatment, all beg for novel antivirals. However, new methods and platforms are required to increase the chances for their discovery.\ud
The classical secretory pathway is essential for biological functions and is utilized for the transport of proteins to the cell surface and/or extracellular matrix. Within the secretory pathway resides an array of enzymes that modify proteins into their mature and active forms. Viruses, as well as other pathogens, hijack such enzymes for their own benefit. While HIV and DenV are distant viruses, both rely on the cellular protease Furin within this compartment for maturation of their viral envelopes. Importantly, blockade of Furin processing leads to non-fusogenic virions, making it an ideal drug target.\ud
In order to monitor proteolysis within the natural milieu of the secretory pathway, an assay with a robust and quantitative read-out was developed. The assay relies on a complex scaffold molecule targeted to the Endoplasmic Reticulum for transport to the cell surface. A substrate is flanked by the FLAG and HA tags fused to the N-terminus of the scaffold. In such a way, in the absence of proteolysis both tags are presented on the cell surface but only one tag (HA) is presented if proteolysis occurs. Tag presentation can be subsequently detected by fluorescent-coupled antibodies in a robust and quantitative manner through flow cytometry and/or microscopy techniques.\ud
The assay described has been adapted to monitor processing of the HIV envelope and DenV pr-M proteins. It has been further calibrated in a 96-well plate format for flow cytometry, to demonstrate both robustness and repeatability, critical for high throughput screening. In addition, varied intensities of a stably expressed fluorescent protein were used for genetic barcoding through retroviral technology. Genetic barcoding was exploited to further develop the platform in a multiplexed format to assay multiple substrates in the same well. A robust platform such as the one described here will expand high throughput capabilities in search for novel antivirals and can additionally be adapted to other substrates in the secretory pathway
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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