Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Abstract

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.Polyomavirus medium-T antigen, as well as the polyomavirus and simian virus 40 (SV40) small-t antigens (small-t), form complexes with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase (31, 49) consisting of three subunits, the catalytic subunit and two regulatory subunits, A and B (11). To elucidate the role of these complexes in neoplastic transformation by polyomavirus and SV40, it is important to determine the effect of T-antigen binding on the enzymatic properties of PP2A. In the accompanying article (50), we demonstrate that purified SV40 small-t binds to the free A subunit and the AC form of PP2A but not to the free C subunit or the ABC form. We also show that small-t inhibits dephosphorylation of myelin basic protein and myosin light chains by the AC form but has no effect on the activity of free C or the ABC form. With phosphorylated histone Hi as an exogenous substrate, small-t stimulates the activity of the AC form. The B subunit and small-t appear to bind to the same site on AC and have similar effects on its activity.In this article, we investigated the substrate specificity of PP2A towards SV40 large-T antigen and the cellular protein p53 and how small-t alters this activity. Large-T is phosphorylated at eight or more sit...

Nuclear protein phosphorylation and growth control

“…Similar observations have been made by others (Sturzbecher et al, 1987). Of additional interest is the finding that the SV40 small t antigen may promote phosphorylation of p53 by blocking protein phosphatase 2A-dependent dephosphorylation of p53 (Scheidtmann et al, 1991b). Taken (Meisner & Czech, 1991 Nuclear protein phosphorylation and growth control Answers to these questions will undoubtedly provide a better understanding about key areas of growth control such as differentiation, development and cellular transformation.…”

“…Concomitant activation of cdkl and PP2A at G1/S could therefore switch T antigen into an active replicating protein in a cell-cycle-dependent manner, thus allowing exploitation of the cell's replication machinery during S phase. It is also possible that the small t antigen of SV40, which down-regulates the activity of PP2A towards T antigen at the inhibitory phosphorylation sites (Yang et al, 1991;Scheidtmann et al, 1991b), is necessary to maintain T antigen in an inactive replication state until its activation at an appropriate point in the cycle. Prior to S phase, T antigen may be employed in other function(s) such as transcription or tumour suppressor protein binding.…”

Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its transcriptional and apoptotic activities

“…At the p53 N-terminus, there are 10 serine/threonine residues (Ser-6, -9, -15, -20, -33, -37, -46, and , PP2A (Scheidtmann et al, 1991), PP5 (Zuo et al, 1998), Wip1 and Cdc14 (Fiscella et al, 1997;Li et al, 2000), can dephosphorylate p53, there is no definitive assignment of the concrete phosphatase responsible for dephosphorylating the specific serine/threonine sites until recently. Long et al The inducibility of the Tet-on system.…”

Involment of p53, Bax, and Bcl‐2 pathway in microcystins‐induced apoptosis in rat testis

“…Thus, p53 is responsible for regulating of cell death through Bax/Bcl-2 imbalances. Furthermore, p53 is a phosphoprotein, it has been shown that in vivo phosphorylated p53 can be dephosphorylated in vitro by PP2A (Scheidtmann et al, 1991). Besides, it has been found that PP2A inhibitor can induce hyperphosphorylation of p53 and increased apoptosis (Yan et al, 1997;Milczarek et al, 1999).…”