Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity.

Abstract

Middle T antigen of polyoma virus is associated principally with the plasma rmembrane. Comparison of the trvpsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [y-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32P04were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

“…Unlike with the other viral oncogenes, however, the associated kinase activity of MTag does not appear to be a property of the antigen itself, because MTag synthesized in Escherichia coli and MTag synthesized in vitro do not possess enzymatic activity (37,38). Moreover, MTag does not possess ATP-binding activity (38).…”

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).…”

Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

“…The protein has an associated kinase activity (9,25,27,29) which phosphorylates protein tyrosine residues (1,9) and phosphatidylinositol [M. Whitman, D. Kaplan, B. Schaffhausen, L. Cantley, and T. M. Roberts, Nature (London), in press]. However, middle T appears to lack intrinsic kinase activity (24,25,28). pp60csrc is associated with middle T (1,7,8) and is apparently the source of the associated kinase activity.…”

“…It is a membranebound protein anchored by a stretch of hydrophobic amino acids at its C terminus (4,12,28). There are two forms of middle T, a predominant 56,000-molecular-weight (56K) species and a minor 58K species (23,24) differing in their serine and threonine phosphorylation.…”