Polyomavirus middle T antigen downregulates junctional cell-to-cell communication.

Azarnia, R.; Loewenstein, Werner R.

doi:10.1128/mcb.7.2.946

Cited by 58 publications

(26 citation statements)

References 20 publications

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“…In contrast, v-src redirected to other subcellular locations was either mitogenic or benign (30a). Both mT and v-src have been proposed to interfere with cell-to-cell communication at adherens junctions (2,3). Adherens junctions are one of the areas in the cell where the cytoskeleton and membrane skeleton are joined (46).…”

Section: Discussionmentioning

confidence: 99%

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

Andrews¹,

Gupta²,

Abisdris³

1993

Mol. Cell. Biol.

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The transforming protein of polyomavirus, middle T antigen, is associated with cellular membranes. We have examined the subcellular location of the middle T antigen in two different cell types by fractionation and detergent phase partitioning. Middle T antigen expressed in human cells by a recombinant adenovirus was detected primarily in the membrane skeleton. Sucrose gradient fractionation revealed that the middle T antigen was associated with complexes with molecular weights of 500,000 to 1,000,000. Several markers for cytoskeleton cofractionate with these complexes, including actin, tubulin, and vimentin. Electron micrographs of membrane skeleton prepared from cells expressing middle T antigen demonstrated that this material contained primarily fibrous structures and was clearly devoid of bilayer membranes. These structures were distinct from the filamentous structures observed in fractions enriched for cytoskeleton. Consistent with a role for membrane skeleton localization in transformation, middle T antigen was detected exclusively in fractions enriched for membrane skeleton in middle T antigen-transformed Rat-2 cells. Our results may resolve the apparent difference between middle T antigen localization as determined by immunomicroscopy and that determined by subcellular fractionation.The transforming protein of polyomavirus is the middle T antigen (mT). Analysis of a variety of cell lines and transgenic animals has demonstrated that mT is sufficient to transform both established cell lines and a wide variety of tissues (1,39,49,51,54). Mutational analyses suggest that the interaction of mT with cellular proteins is critical for transformation. In polyomavirus-infected cells, established cell lines expressing mT, and 293 cells infected with a recombinant adenovirus vector, mT has been shown to interact with members of the src family of tyrosine kinases (11,15,25,29,30,35,38), phosphoinositol-3-kinase (PI-3-kinase) (12, 19, 32, 36, 52), phosphatase 2A (22, 37, 50), and other as-yet-unidentified polypeptides. Complex formation with at least one member of the src family as well as with PI-3-kinase is necessary but not sufficient for transformation in vitro (11,52). Association of mT with these molecules appears to activate both kinases (7,45). Moreover, it appears that accumulation of the phosphorylated products of the PI-3-kinase is required for transformation (32). Nevertheless, only a fraction of mT molecules in cells are complexed to either the src family kinases or PI-3-kinase; therefore, it remains possible that additional interactions are involved in cellular transformation (23).Subcellular fractionation, immunofluorescence, and immunoelectron microscopy of mT-expressing cells suggest that mT is found in association with subcellular membranes (4,9,18,41,44,47,55). It has been assumed that mT is an integral membrane protein because of a contiguous 22-amino-acid hydrophobic segment near the carboxyl end of the molecule. Mutations in this region of mT abrogate both membrane association and transformation (...

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Section: Discussionmentioning

confidence: 99%

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

Andrews¹,

Gupta²,

Abisdris³

1993

Mol. Cell. Biol.

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“…Reduced GJC is a characteristic of cells transformed by several oncogenes (24), including SV-40 (33), polyomavirus middle T antigen (34), v-ras (35,36), v-mos (37), and v-fps (38), as well as v-src. The effects of Src on GJC have been studied extensively (17-20, 30, 32, 39 -41).…”

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Tyrosine Phosphorylation of Connexin 43 by v-Src Is Mediated by SH2 and SH3 Domain Interactions

Kanemitsu

Loo

Simon

et al. 1997

Journal of Biological Chemistry

185

160

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Reduction of gap junctional communication in v-The Rous sarcoma virus transforming protein, v-Src, and its cellular homologue, c-Src, are protein tyrosine kinases associated with the plasma membrane (1). Expression of v-Src leads to neoplastic cell transformation, accompanied by increased tyrosine phosphorylation of cellular proteins (2-4). Signaling through Src is due, in part, to protein-protein interactions mediated by SH2 1 and SH3 domains (5-7). Mutations in either the SH2 or SH3 domain of c-Src can lead to increased tyrosine kinase activity and oncogenic potential (5,8,9). SH2 domains bind Tyr(P) residues in target proteins. The specificity of binding is determined by residues immediately C-terminal to the Tyr(P) (6, 10, 11). SH3 domains bind short proline-rich peptide motifs (6,(12)(13)(14). Studies using phage display libraries to identify peptides that bind SH3 domains showed that a minimal PXXP consensus sequence is required for binding (15,16).In addition to playing a role in cell growth and transformation, the Src protein tyrosine kinase has been implicated in regulating GJC (17)(18)(19)(20). Gap junctions are membrane channels which mediate the intercellular passage of ions, second messengers, and small molecules (21). It has been proposed that growth regulatory molecules pass between cells through gap junctions (22)(23)(24)(25).Gap junctions are formed by specialized proteins termed connexins, arranged in the cell membrane so that each connexin has four membrane spanning regions, a cytoplasmic loop, two extracellular loops, and cytoplasmic N-and C-terminal ends. Among the 13 connexin family members identified to date, the C-terminal tail is the most divergent region. In some cases this region contains consensus protein kinase phosphorylation sites (26 -28). Connexins 32 and 43 are phosphoproteins (29 -32), however, Cx43 is phosphorylated on tyrosine by Src, but Cx32 is not (32). Comparison of the amino acid sequences of connexins 32 and 43 revealed that putative SH3-binding regions and tyrosine phosphorylation sites in Cx43 were absent in connexin 32 (26).Reduced GJC is a characteristic of cells transformed by several oncogenes (24), including SV-40 (33), polyomavirus middle T antigen (34), v-ras (35, 36), v-mos (37), and v-fps (38), as well as v-src. The effects of Src on GJC have been studied extensively (17-20, 30, 32, 39 -41). Several lines of evidence suggest that tyrosine phosphorylation of Cx43 is important in the regulation of GJC by v-Src. First, cells infected with temperaturesensitive Rous sarcoma virus show reduced levels of GJC (17,19), correlated with a rapid increase in tyrosine phosphorylation of Cx43 (40), at the permissive temperature. Second, expression of v-Src in communication-competent paired Xenopus oocytes expressing Cx43, but not in oocytes expressing Cx32, leads to reduced GJC, which depends on phosphorylation of Cx43 on tyrosine 265 (32). Finally, purified Src phosphorylates Cx43 in vitro at sites that are phosphorylated in vivo in v-src transformed Rat-1 fibroblasts (41)...

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“…Furthermore, fibroblasts infected with wildtype RSV or transfected with v-src or c-src gene constructs containing kinase-activating mutations exhibited reductions in gap-junctional communication (4,6,14; D. Crow and A. Lau, unpublished observations). Likewise, kinase-active pp60c-sr( associated with polyomavirus middle T antigen decreased rat F cell junctional permeability (5,10,17). pp60v-src has also been localized to membrane regions containing gap junctions and other intercellular junctions (36).…”

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Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Crow¹,

Beyer²,

Paul³

et al. 1990

Mol. Cell. Biol.

332

255

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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 pp6Ov-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.

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Polyomavirus middle T antigen downregulates junctional cell-to-cell communication.

Cited by 58 publications

References 20 publications

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

Tyrosine Phosphorylation of Connexin 43 by v-Src Is Mediated by SH2 and SH3 Domain Interactions

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

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