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Item Description Crow, David Scott en_US 2009-07-15T17:16:30Z 2009-07-15T17:16:30Z 1990 en_US
dc.description Typescript. en_US
dc.description Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. en_US
dc.description Includes bibliographical references (leaf 80) en_US
dc.description Microfiche. en_US
dc.description viii, 80 leaves, bound ill. 29 cm en_US
dc.description.abstract Gap junctions are membrane channels permitting interchange of ions and small molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60^v-src action. This dissertation investigates this possibility by identifying and characterizing putative fibroblast proteins (connexins) involved in junctional communication. Findings indicate that uninfected mammalian fibroblasts do not contain RNA or protein related to liver connexin32. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kb RNA similar in size to connexin43 RNA in heart tissues. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43-kD, 45-kD and 47-kD from communicating fibroblasts. Gap junctions from heart cells contained predominantly 45-kD and 47-kD species similar to those in fibroblasts. Uninfected fibroblast 45-kD and 47kD proteins were phosphorylated on serine residues. Phosphatase digestions of 45-kD and 47-kD fibroblast and heart connexin proteins indicated that they represent phosphorylated forms of the 43-kD protein. Additionally, proteolytic mapping of the fibroblast and heart connexin proteins showed them to have similar primary structures. Pulse-chase labeling studies showed that phosphorylation of the fibroblast connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In contrast to these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlated with the loss of gap-junctional communication in RSV-transformed fibroblasts. Furthermore, non-transformed cells expressing kinase-active, non-myristylated pp60^2A527F communicated at high levels and contained connexin43 phosphorylated predominately on serine, but not tyrosine. In contrast, cells transformed by kinase-active, myristylated pp60^527F did not communicate and contained connexin43 proteins phosphorylated on serine and tyrosine. Additionally, activation of pp60v-src, temperature sensitive for transformation, resulted in a rapid, simultaneous increase in relative phosphotyrosine content of connexin43 protein and loss of gap-junctional communication. These combined results suggested that connexin43 is a functionally-relevant substrate of pp60^v-src whose phosphorylation on tyrosine may be involved in the disruption of gap-junctional communication. en_US
dc.language.iso en-US en_US
dc.relation Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Biomedical Sciences (Biochemistry); no. 2543 en_US
dc.rights All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner. en_US
dc.subject Fibroblasts en_US
dc.subject Rous sarcoma en_US
dc.subject Connective tissue cells en_US
dc.subject Intermediate filament proteins en_US
dc.title Identification and characterization of gap junction-associated proteins phosphorylated in RSV-infected fibroblasts en_US
dc.type Thesis en_US
dc.type.dcmi Text en_US

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