Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N-and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.The transforming proteins of small DNA tumor viruses form multiple complexes with cellular proteins involved in signal transduction and growth control. These interactions play an important role in virus-induced tumorigenesis. Simian virus 40 (SV40) large T binds to the tumor suppressor proteins p53 (40, 42) and Rb (16) and presumably inactivates their function. Polyomavirus medium T associates with pp6Oc-src and activates its protein-tyrosine kinase activity (4,12,14). It also binds to other members of the c-src family (9,36,39). In addition, medium T binds to and activates phosphatidylinositol-3 kinase (13, 32). Moreover, medium T forms a complex with two cellular proteins of approximately 61 and 37 kDa (23,24,35,50,59,60,64). The two proteins are associated with each other in uninfected cells (23). The medium T antigen of nontransforming hrt mutants (3) does not form a complex with the 61-kDa and 37-kDa proteins but binds to the 73-kDa heat shock protein instead (23,24,35,50,60,64,70). These data suggest that complex formation between medium T and the 61-kDa and 37-kDa proteins might be necessary for transformation. SV40 small T forms a complex with two cellular proteins of approximately 56 and 32 kDa (76). These proteins are also associated with polyomavirus and BK virus small T (55, 56). They are identical to the medium-T-associated 61-kDa and 37-kDa proteins, respectively (50, 71).The 61-kDa protein was purified and partially sequenced, and its cDNA was cloned from a human cDNA library (72). Its predicted amino acid sequence revealed a protein consisting of 15 imperfect repeats, most of which are 39 amino acids long. It had no resemblance to known proteins in data banks. The 37-kDa protein was also purified, partially se-* Corresponding author. t Present address:

Protein phosphatase 2A consists of three subunits, the catalytic subunit (C) and two regulatory subunits (A and B). The A subunit has a rod-like shape and consists of 15 nonidentical repeats. It binds the catalytic subunit through repeats 11 to 15 at the C terminus and the tumor antigens encoded by small DNA tumor viruses through overlapping but distinct regions at N-terminal repeats 2 to 8. A model of the A subunit was developed on the basis of the fact that uncharged or hydrophobic amino acids are conserved at eight defined positions within each repeat. Helical wheel projections suggested that each repeat can be arranged as two interacting amphipathic helixes connected by a short loop. Mutational analysis of the A subunit revealed that the proposed loops are important for binding of tumor antigens, the B subunit, and the C subunit. Native gel analysis of mutant A subunits synthesized in vitro demonstrated that the binding region for the B subunit, previously thought to include repeats 2 to 8, covers repeats 1 to 10 and that the B and C subunits cooperate in binding to the A subunit.

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.