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Selective ability of S-phase cell extracts to dephosphorylate SV40 large T antigen in vitro
1University of Rochester Cancer Center, Division of Tumor Biology, New York 14642.
Abstract:
Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.
Insights
Simian virus 40 (SV40) large tumor antigen (T) dephosphorylation by protein phosphatase 2A (PP2A) is cell cycle-dependent. This specific phosphatase activity is highest during the S phase, coinciding with host-cell DNA synthesis.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein regulated by phosphorylation.
- Protein phosphatase 2A (PP2A) dephosphorylates SV40 T-antigen, activating viral DNA replication.
- The cell cycle regulation of PP2A activity towards T-antigen remains unclear.
Purpose of the Study:
- To investigate the cell cycle-specific regulation of PP2A activity on SV40 T-antigen.
- To determine how PP2A specificity towards T-antigen is controlled during the cell cycle.
Main Methods:
- Developed an in vitro assay to measure T-antigen dephosphorylation.
- Used 32P-labeled, immunocomplexed T-antigen as a substrate.
- Incubated labeled T-antigen with cell extracts from different cell cycle phases (G1, S, M).
- Compared T-antigen dephosphorylation with a control substrate (phosphorylase a).
Main Results:
- Cell extracts from S-phase cells selectively dephosphorylated T-antigen compared to G1 and M phases.
- T-antigen specific phosphatase activity was detected at the onset of host-cell DNA synthesis.
- PP2A activity towards phosphorylase a was observed throughout the cell cycle, indicating substrate specificity.
Conclusions:
- PP2A activity towards SV40 T-antigen is regulated in a cell cycle-dependent manner.
- This regulation is specific to T-antigen and not a general phosphatase activity.
- The findings suggest a mechanism linking viral replication to the host cell cycle machinery.