MITOSIS-SPECIFIC PHOSPHORYLATION OF POLYOMAVIRUS MIDDLE-SIZED TUMOR-ANTIGEN AND ITS ROLE DURING CELL-TRANSFORMATION

Transformation of cells in culture by polyomavirus is mediated by one of its early gene products, middle-sized tumor antigen (MTAg). This pr otein forms multiple complexes with cellular enzymes such as tyrosine kinases (pp60c-src), a phosphatidylinositol 3-kinase, and phosphatase 2A. Association with MTAg leads to the activation of pp60c-src through interference with phosphorylation at Tyr-527, a site negatively regul ating src kinase activity. MTAg abrogates mitosis-specific activation of pp60c-src, resulting in constitutive high kinase activity of the en zyme throughout all phases of the cell cycle. Here we report that MTAg is transiently modified during mitosis, resulting in an increase in i ts apparent molecular size on SDS/acrylamide gels. Similarly, MTAg iso lated from interphase cells and phosphorylated by the cell cycle-regul ated serine/threonine kinase p34cdc2 in vitro has increased molecular mass. The large molecular mass form of the protein can be converted to the authentic 56-kDa form upon dephosphorylation by potato acid phosp hatase. Two putative phosphorylation sites for a cdc2-like kinase were identified as Thr-160 and -291, respectively. Conversion of Thr-160 t o Ala resulted in a transformation-defective mutant protein that was s till capable of associating with pp60c-src, phosphatidylinositol 3-kin ase, and phosphatase 2A, while the corresponding mutant in position 29 1 was wild type with respect to all parameters measured so far. These data suggest that phosphorylation by p34cdc2 or a related cell cycle-r egulated kinase modulates the interaction of MTAg with cellular target s that are crucial for cell transformation.