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Identification and characterization of gap junction-associated proteins phosphorylated in RSV-infected fibroblasts
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|Title:||Identification and characterization of gap junction-associated proteins phosphorylated in RSV-infected fibroblasts|
|Authors:||Crow, David Scott|
Connective tissue cells
Intermediate filament proteins
|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.|
Thesis (Ph. D.)--University of Hawaii at Manoa, 1990.
Includes bibliographical references (leaf 80)
viii, 80 leaves, bound ill. 29 cm
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Ph.D. - Biomedical Sciences (Biochemistry)|
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