Interaction with SV40 small tumor antigen (small t) compromised the ability of multimeric protein phosphatase 2A to inactivate the mitogen-activated protein kinase ERK1 and the mitogen-activated protein kinase kinase MEK1. Transient expression of small t in CV-1 cells activated MEK and ERK but did not affect Raf activity. Small t stimulated the growth of quiescent CV-1 cells almost as effectively as did serum. Coexpression of kinase-deficient ERK2 blocked most, but not all, of the proliferation caused by small t. Activation of the mitogen-activated protein kinase pathway and stimulation of cell growth were dependent on the interaction of small t with protein phosphatase 2A. These findings indicate that SV40 small t is capable of inducing cell growth through blockade of protein phosphatase and deregulation of the mitogen-activated protein kinase cascade.

Initiation of DNA replication in eukaryotes is dependent on the activity of protein phosphatase 2A (PP2A), but specific phosphoprotein substrates pertinent to this requirement have not been identified. A novel regulatory subunit of PP2A, termed PR48, was identified by a yeast two-hybrid screen of a human placental cDNA library, using human Cdc6, an essential component of prereplicative complexes, as bait. PR48 binds specifically to an amino-terminal segment of Cdc6 and forms functional holoenzyme complexes with A and C subunits of PP2A. PR48 localizes to the nucleus of mammalian cells, and its forced overexpression perturbs cell cycle progression, causing a G 1 arrest. These results suggest that dephosphorylation of Cdc6 by PP2A, mediated by a specific interaction with PR48, is a regulatory event controlling initiation of DNA replication in mammalian cells.Initiation of chromosomal DNA replication in eukaryotes is a multistep process that requires assembly of prereplicative complexes on chromosomal DNA, followed by unwinding of replication origins and loading of replicative polymerases and accessory proteins that catalyze DNA synthesis (reviewed in references 6, 34, and 35). Stringent control mechanisms, directed by the competing activities of multiple protein kinases and protein phosphatases, ensure that each segment of the genome is replicated once and only once in each cell cycle and that DNA replication is properly timed with respect to cell growth and mitosis.The serine/threonine protein phosphatase 2A (PP2A) exerts regulatory control over the initiation of DNA replication in yeast, viral, and vertebrate systems (19, 20, 33). This relationship was demonstrated first for replication of simian virus 40 (SV40) DNA, where dephosphorylation of specific serine residues by PP2A within large T antigen promotes DNA binding of T-antigen hexamers and unwinding of SV40 replication origins (33). More recently, immunodepletion of PP2A holoenzyme was observed to prevent chromosomal DNA replication in a Xenopus cell-free replication system (20). Neither assembly of prereplicative complexes nor elongation of replication forks is dependent on PP2A, but firing of replication origins is suppressed in the absence of this enzyme. This requirement for PP2A activity in promoting DNA replication presumably is based on direct or indirect modification of the proteins responsible for priming and/or firing replication origins. However, with the exception of SV40 large T antigen, specific phosphoprotein substrates of PP2A that are pertinent to its role in promoting DNA replication have not been identified.Cdc6 proteins are highly conserved in organisms as diverse as Saccharomyces cerevisiae, Xenopus laevis, and Homo sapiens (1,4,8,22,31,41) and exert unique functions at each of several steps of replication initiation. Cdc6 is required for formation of prereplicative complexes and is rate limiting for initiation of DNA replication in all species in which the function of Cdc6 has been examined (4, 9, 18, 36, 42). In yeast, forced overexpr...

SV40 small tumor antigen (small-t) was used as a model to identify structural elements involved in the interactions between regulatory proteins and protein phosphatase 2A (PP2A). Using mutant proteins and synthetic peptides, we identified a small domain within small-t that is a major site for interaction with the dimeric form of PP2A. A series of small-t truncation mutants identified a region surrounding the first of two conserved cysteine clusters that was critical for interaction with PP2A. These mutants also identified additional regions of small-t that contribute to high affinity interaction. Deletion of residues 110 -119, which encompass the first cysteine cluster, resulted in a protein that failed to bind to PP2A. Synthetic peptides that contained residues 105-122 of small-t blocked binding of small-t to PP2A. These peptides also inhibited the phosphatase activity of PP2A in a manner analogous to fulllength small-t. The active small-t peptides adopt a ␤-strand structure that was essential for high affinity interaction with the PP2A dimer. Based on circular dichroism measurements, the same cysteine cluster-containing peptides that bind to PP2A also interact with zinc. Interaction with zinc required the conserved cysteines but was not required for interaction with PP2A.

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