It is clear that much remains to be discovered regarding the roles of protein phosphatases in mitogenic signaling pathways. The ability of okadaic acid to activate MAPK/ERKs demonstrates that alteration in serine/threonine dephosphorylation can have significant effects on common steps in growth stimulation induced by different types of mitogens. As in the case of cell cycle control, protein serine/threonine phosphatase plays a central role in the reentry of quiescent cells into the cycle. Because the only known targets of okadaic acid are the catalytic subunits PP1 and PP2A, these enzymes are crucial components of two basic functions carried out by cells: growth and division. Important and obligatory roles for PP2B, PP2C, and newly discovered serine/threonine phosphatases are also likely. However, the limited tissue distribution, unique regulatory properties, and limited substrate specificities of these forms suggest more specialized functions in restricted cell types. The available information on the specific functions of different forms of protein serine/threonine phosphatases, let alone their individual isoforms and different multimeric holoenzymes, is still severely limited. Years of biochemical characterization and cDNA cloning have left us with far more forms than functions. This has led to the gratifying situation, at least for the biochemists, in which genetics and cell biology identify protein phosphatases for which a wealth of biochemical information is already available. The appreciation of the importance of these enzymes in the coming years can only increase as the functions for individual forms are discovered.

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity.Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Smail-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone Hi phosphorylated by protein kinase C. With the exception of histone HI, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone Hi, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone Hi as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-t antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.Neoplastic transformation by polyoma-, papilloma-, and adenoviruses involves complex formation between their transforming proteins and cellular proteins involved in regulating cell proliferation (20). For example, the transforming protein of simian virus 40 (SV40), large T antigen (large T), binds to and presumably inactivates the growth-suppressing proteins p53 (see reference 30 for review) and the product of the retinoblastoma gene (RB) (17,18). The transforming proteins of some human papillomaviruses also form complexes with p53 and the RB protein, suggesting a similar mechanism of transformation. On the other hand, the principal transforming protein of polyomavirus, medium-T antigen (medium T), does not bind to p53 or the RB protein.Instead, medium T associates with pp60csrc, the product of the c-src proto-oncogene (14-16), and strongly activates its protein-tyrosine kinase activity (4,9,14). Genetic evidence indicates that the activation of pp60csrc plays a role in transformation by polyomavirus (3,16). Medium T also binds to the pp62c-Yes (27) and pp59C-fy (10, 28) proteins, two pp60CcSrc_relat...

Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-ArgSer-Arg-His-Phe (KF), and an internal region, Glu-Glu-GluGlu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp6OCSrC and a new protein of "61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of =72,000 Da.The transforming protein of polyoma virus, medium T antigen, is a membrane protein (1-3) associated with a tyrosine-specific protein kinase activity (4-6). Biochemical studies have indicated that this activity is not intrinsic to medium T antigen (7,8) but instead resides in a minor fraction sedimenting with a size of 200,000 Da, whereas the bulk of medium T antigen is monomeric and kinase inactive (9). It was shown subsequently that the kinase activity is due to an association between medium T antigen and pp6O1(src, the cellular homolog of the transforming protein of Rous sarcoma virus, pp6OVSrC (10). The interaction between medium T antigen and pp60'-sr may be facilitated by the fact that both proteins are membrane associated and, therefore, come into close contact. In a similar fashion, medium T antigen might interact with other neighboring membrane proteins. We asked how one might identify such proteins without having a functional assay or a specific antiserum available. In the present paper, we demonstrate that in addition to pp60O-s>, medium T antigen also forms a complex with a cellular protein of -61,000 Da. This was achieved by affinity chromatography with two anti-peptide antibodies directed against different regions of medium T antigen.

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:

The polyoma virus medium and small tumor antigens, as well as simian virus 40 small tumor antigen, form specific complexes with two cellular proteins designated 61-and 37-kDa proteins. In this report, we demonstrate that the 61-and 37-kDa proteins correspond to the A and C subunits, respectively, of the serine-and threonine-specific protein phosphatase 2A (PP2A). On the one hand, antibodies raised against the 61-kDa protein reacted specifically with the purified A subunit of PP2A. Furthermore, the amino acid sequences of seven tryptic peptides from the A subunit were almost identical to sequences of the 61-kDa protein as deduced from the corresponding cDNA sequence. On the other hand, antibodies against the purified C subunit (catalytic subunit) of PP2A reacted specifically with the medium tumor antigen-associated 37-kDa protein. These data suggest a role of PP2A in cell transformation by polyoma virus and sinmian virus 40.The importance of protein phosphorylation in signaling pathways that control cell proliferation and carcinogenesis is well established (for review, see ref. 1). Many oncogenes and growth factor receptors are tyrosine-specific protein kinases and two oncogenes, mos and raf, are serine/threoninespecific protein kinases (2,3). Little is known about the role of protein dephosphorylation in growth control, although it seems likely that reversible regulatory mechanisms involving specific phosphorylation events also require specific mechanisms for dephosphorylation. It has recently been shown that several tyrosine-specific protein phosphatases comprise a family ofreceptor-linked enzymes, which may be important in attenuating the effects of tyrosine phosphorylation on cell proliferation (for review, see ref. 4). Four major forms of serine/threonine-specific phosphatases have been characterized in mammalian tissues, which are differentiated by substrate specificity, sensitivity to inhibitors, regulatory properties, antibody reactivity, and primary sequence (for review, see ref. 5). Protein phosphatase 2A (PP2A) comprises a significant portion of total activity in many tissues, has a broad substrate specificity, and is active in the absence of divalent cations. It consists of a 36-to 38-kDa catalytic subunit (termed C) and two additional subunits of 60 and 55 kDa, termed A and B, respectively (5). cDNA clones encoding the catalytic subunit have been isolated from a number of tissues and species (6-8). Analysis of the primary amino acid sequence has shown that the type 2A catalytic subunit is highly conserved between species (nearly 100o identity) and that it is a member of a gene family that includes the catalytic subunit of types 1 and 2B protein phosphatases (5, 9). Currently, very little is known about the structure or function of the A and B subunits. The presence or absence of the A, or A and B, subunits alters the activity and substrate preference of the catalytic subunit (10-12).The transforming proteins of DNA tumor viruses frequently form complexes with cellular proteins. These interact...