PHOSPHATASE 2A ASSOCIATED WITH POLYOMAVIRUS SMALL-T OR MIDDLE-T ANTIGEN IS AN OKADAIC ACID-SENSITIVE TYROSYL PHOSPHATASE

Papovavirus tumor antigens have been shown to associate with the cellu lar phosphoserine/threonine-specific protein phosphatase 2A (PP2A). We were interested in the consequences that T-antigen association might have on PP2A activity and so studies of the phosphatase activity in im munoprecipitates, prepared from polyoma virus-transformed or polyoma v irus-infected mouse 3T3 fibroblasts, were performed. The phosphoserine /threonine phosphatase activity, measured with phosphorylase a as the substrate, showed all the characteristics of PP2A. It was stimulated b y polycations, inhibited by fluoride or p-nitrophenyl phosphate, sensi tive to okadaic acid and microcystin and insensitive to inhibitor-1 an d inhibitor-2. Phosphotyrosyl phosphatase (PTPase) activity was associ ated with the middle-T/small-T-associated complex when reduced, carbox amidomethylated and maleylated lysozyme, phosphorylated exclusively on tyrosyl residues, was used as the substrate. This PTPase activity was as sensitive to okadaic acid as was the phosphorylase phosphatase act ivity; it could be inhibited by phosphorylase a and did not dephosphor ylate poly(Glu80Tyr20). The level of middle-T/small-T-associated PTPas e activity relative to the phosphorylase phosphatase activity was tenf old higher than that of the purified dimeric PP2A. A similar activity ratio was observed with the purified phosphatase after stimulation wit h a cellular protein, designated phosphotyrosyl phosphatase activator. These results suggest that the same enzyme may possess dual specifici ty. In contrast to the cellular trimeric PP2A, containing the 55-kDa p utative regulatory subunit, the middle-T/small-T-associated enzyme had low activity towards a retinoblastoma peptide phosphorylated by p34cd c2. These results indicate how middle-T/small-T might effect the activ ity of PP2A in polyoma virus-transformed cells.