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

Abstract

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 (...

At the onset of transformation polyomavirus middle-T recruits shc and src to a perinuclear compartment coincident with condensation of endosomes

“…Although extensively studied, the site of mT membrane localization remains unresolved with morphologic studies favouring intracellular membranes (Dilworth et al, 1986;Templeton et al, 1984;Zhu et al, 1984) while biochemical analyses continue to be interpreted as suggesting that kinase active mT containing complexes are located at the plasma membrane (Ballmer-Hofer and Benjamin, 1985;Ito et al, 1977;Scha hausen et al, 1982;Segawa and Ito, 1982). Recently, we have demonstrated a role for cytoskeletal proteins, particularly actin, in the localization of mT (Andrews et al, 1993). These results have been con®rmed and extended to suggest that mT may interact with the cytoskeleton at more than one location (Krauzewicz et al, 1994).…”

“…Using similar methods, the P100 fraction of Ad5/mT infected MRC5 cells was fractionated by solubilization with non-ionic detergent at low ionic strength and centrifugation to pellet the cytoskeleton (C). Phase partitioning of the remaining soluble material yielded a fraction containing short actin ®laments that was previously assumed to originate primarily from the sub-membraneous cytoskeleton (M), as well as fractions containing aqueous soluble proteins (mostly lumenal contents) (A) and hydrophobic membrane proteins (D) (Figure 10b) (Andrews et al, 1993). At 40 h post-infection with Ad5/mT, shc was recovered almost exclusively in the fraction containing cytoskeleton (Figure 10b).…”

“…Previously, we demonstrated that mT can be released from P100 fractions prepared from late stage Ad5/mT infected 293 cells and from transformed rat ®broblasts in large complexes containing actin (Andrews et al, 1993). Using similar methods, the P100 fraction of Ad5/mT infected MRC5 cells was fractionated by solubilization with non-ionic detergent at low ionic strength and centrifugation to pellet the cytoskeleton (C).…”

“…However, the role of mT in transformation has been determined almost exclusively in transformed or lytically infected cells and at a single time point (Andrews et al, 1993;Carmichael et al, 1982;Dilworth et al, 1986;Krauzewicz et al, 1994;Segawa and Ito, 1982;Zhu et al, 1984). As a result, little is known about the initial changes that occur in cells expressing mT.…”

“…The protein is usually described as a membrane protein without intrinsic enzymatic activity (Andrews et al, 1993;Gri n and Dilworth, 1983;Kaplan et al, 1988). Although extensively studied, the site of mT membrane localization remains unresolved with morphologic studies favouring intracellular membranes (Dilworth et al, 1986;Templeton et al, 1984;Zhu et al, 1984) while biochemical analyses continue to be interpreted as suggesting that kinase active mT containing complexes are located at the plasma membrane (Ballmer-Hofer and Benjamin, 1985;Ito et al, 1977;Scha hausen et al, 1982;Segawa and Ito, 1982).…”