RESUMO
Disruption of gap junctional communication (measured by intercellular dye transfer) in cultured fibroblasts by pp60v-src is correlated with phosphorylation of the gap junction protein, connexin43 (cx43), on tyrosine. In this report, we examine the functional relevance of these observations by studying cx43 phosphorylation in cells containing kinase-active, non-myristylated pp60(2A527F) or pp60v-src temperature sensitive (ts) for transformation. Non-transformed cells expressing pp60(2A527F) transferred fluorescent dye at high levels and contained cx43 that was phosphorylated predominately on serine. In contrast, cells transformed by kinase-active, myristylated pp60(527F) did not transfer dye and contained cx43 proteins which were phosphorylated on serine and tyrosine. Additionally, activation of ts pp60v-src tyrosine kinase activity upon shift of cells to the permissive temperature was correlated with a rapid increase in the phosphorylated tyrosine content of cx43 proteins and loss of gap junctional communication. These combined results suggested that cx43 is a substrate of pp60v-src whose phosphorylation on tyrosine may be involved in the disruption of gap junctional communication observed in Rous sarcoma virus (RSV)-transformed cells.
Assuntos
Comunicação Celular/fisiologia , Genes src/fisiologia , Proteínas de Membrana/metabolismo , Animais , Transformação Celular Neoplásica/patologia , Conexinas , Técnicas In Vitro , Proteína Oncogênica pp60(v-src)/fisiologia , Fosforilação , RatosRESUMO
Cardiac gap junctions permit the conduction and propagation of the electrical impulses that are responsible for the synchronous contraction of the myocardium (Page and Manjunath, 1986). Cardiac gap junctions are formed by the association of connexin molecules within the plasma membrane of heart cells. They create aqueous channels that permit exchange of ions and other small molecules between adjacent cells. Intercellular communication via these channels may be regulated by phosphorylation. cAMP was shown to increase junctional conductance and stimulate phosphorylation of connexin32 in cultures of dissociated liver hepatocytes (Saez et al., 1986). Furthermore, a 47 kDa protein purified from dog heart gap junctions was phosphorylated in vitro by the catalytic subunit of protein kinase A (Pressler and Hathaway, 1987). Other studies have demonstrated that cAMP enhanced junctional conductance in intact heart and isolated heart cells (De Mello, 1986; De Mello and van Loon, 1987; Burt and Spray, 1988). This report provides direct evidence that the heart gap junction protein, connexin43, from unstimulated heart tissues and cultured myocytes is phosphorylated stoichiometrically in vivo. Phosphorylation of 45 and 47 kDa connexin43-related proteins occurred predominantly on serine. In addition, the 47 kDa protein contained a low-level of phosphothreonine.
Assuntos
Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Fosfatase Ácida , Animais , Células Cultivadas , Conexinas , AMP Cíclico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/isolamento & purificação , Fosforilação , RatosRESUMO
An architecture for providing an institutional systems infrastructure is proposed. The architecture permits distributed applications while maintaining an integrated patient database.
Assuntos
Sistemas Computacionais , Bases de Dados Factuais , Sistemas de Informação Hospitalar , Redes de Comunicação de Computadores , Hospitais Universitários , Microcomputadores , North Carolina , Projetos PilotoRESUMO
Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.