Cell cycle progression in monkey cells expressing simian virus 40 small t antigen from adenovirus vectors
2区 · 医学
作者: Howe, Alan K. ; Gaillard, Stephanie ; Bennett, John S. ; Rundell, Kathleen
The simian virus 40 small t antigen (small-t) is required for optimal viral replication and transformation, especially during the infection of nondividing cells, suggesting that the function of small-t is to promote cell cycle progression. The mechanism through which small-t promotes cell growth reflects, in part, its binding and inhibition of protein phosphatase 2A (PP2A). The use of recombinant adenoviruses allows small-t expression in a majority of cells in a population, thus providing a convenient source of cells for biochemical analyses. In monkey kidney CV1 cells, small-t expressed from these adenovirus vectors activated the mitogen-activated protein kinase (MAPK) pathway, induced JNK activity, and increased AP-1 DNA-binding activity, all in a PP2A-dependent manner. Expression of small-t also caused an increase in the phosphorylation of the Na+/H+ antiporter, a mitogen-activated ion exchanger whose activity correlates with its phosphorylation. At least part of the antiporter phosphorylation induced by small-t reflected activation of the MAPK pathway, as suggested by results of assays using a chemical inhibitor of the MAPK-activating kinase, MEK. Finally, small-t expression from adenovirus vectors promoted efficient cell cycle progression by growth-arrested cells. These vectors should facilitate further analysis of effects of small-t on cell cycle mediators.
1994-03-01·Journal of Virology2区 · 医学
Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation
2区 · 医学
作者: Mungre, Shubhangee ; Enderle, Kristine ; Turk, Ben ; Porras, Analia ; Wu, Yun Qi ; Mumby, Marc C. ; Rundell, Kathleen
Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45.
1991-04-01·Molecular and Cellular Biology3区 · 生物学
Control of protein phosphatase 2A by simian virus 40 small-t antigen
3区 · 生物学
作者: Yang, Sung Il ; Lickteig, Ronald L. ; Estes, Robert ; Rundell, Kathleen ; Walter, Gernot ; Mumby, Marc C.
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. Small-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 H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, 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 H1 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-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.