Control of protein phosphatase 2A by simian virus 40 small-t antigen.

Abstract

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...

Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

“…The SV40ER encodes for LT and ST antigens, which are known to inactivate tumor suppressors such as p53, pRb and PP2A (Yang et al, 1991;Ali and DeCaprio, 2001), and recent studies have identified a role for several oncogenes and tumor-suppressor genes in the regulation of stemness properties (Pardal et al, 2005). For example, downregulation of p53 has been associated with increased selfrenewal, and upregulation of stem cell markers, such as Nanog, ABCB1 and CD44 (Jerry et al, 2008).…”

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen

“…Several lines of evidence suggest that ST perturbs cellular targets that participate in human tumor development (Shenk et al, 1976;Crawford et al, 1978;Choi et al, 1988). The ability of ST to transform human cells requires binding to the abundant serine-threonine protein phosphatase 2A (PP2A) (Yang et al, 1991;Sontag et al, 1993). PP2A is a heterotrimer composed of a conserved catalytic C subunit, a structural A subunit and a regulatory B subunit.…”

Truncated N-terminal mutants of SV40 large T antigen as minimal immortalizing agents for CNS cells

“…While the functions of small t are incompletely understood, in cells that have been subjected to SV40 infection, it is reported to enhance viral replication and polyploidy of the host cells (Whalen et al, 1999) to facilitate transformation of the infected cells by LT in limiting growth conditions (Bikel et al, 1987;Martin and Chou, 1975;Rubin et al, 1982) and to promote induction of cell cycle progression in non-dividing cells, (Porras et al, 1999). Known cellular targets for small t are hsc70 (Srinivasan et al, 1997) and protein phosphatase 2A (Pallas et al, 1990;Yang et al, 1991). Other reported functions of small t include transcriptional activation of promoters of AP-1 (Frost et al, 1994) cyclin A (Porras et al, 1999), and cyclin D1 (Watanabe et al, 1996), repression of c-fos promoters (Wang et al, 1994), and down-regulation of the cdk inhibitor p27 KIP1 (Porras et al, 1999).…”