Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside. To determine whether this MT TMD is inserted into the endoplasmic reticulum (ER) membrane, we added the ER retention signal KDEL to the MT C terminus (MTKDEL). This mutant protein locates only in the ER, demonstrating that MT does insert into membranes solely at this location. In addition, this ER-located MT failed to transform. Examination of the binding proteins associated with the MTKDEL protein demonstrated that it associates with PP2A and c-Src but fails to interact with ShcA, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLC-γ1), despite being tyrosine phosphorylated. Additional mutant and antibody studies show that MT binding to PP2A is probably required for MT to efficiently exit the ER and migrate to the plasma membrane though the TMD also plays a role in this relocation. Overall, these data, together with previous publications, illustrate that MT associates with signaling proteins at different sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the endoplasmic reticulum, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane.
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