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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
Genetic analysis of B55alpha/Cdc55 protein phosphatase 2A subunits: association with the adenovirus E4orf4 protein
Zhiying Zhang1, Melissa Z Mui, Francine Chan
1Key Laboratory of Molecular Biology of Shann'xi Agriculture, College of Animal Science and Technology, Northwest A&F University, 22 XiNong Road, YangLing, Shaan'xi, People's Republic of China.
Abstract:
The human adenovirus E4orf4 protein is toxic in both human tumor cells and Saccharomyces cerevisiae. Previous studies indicated that most of this toxicity is dependent on an interaction of E4orf4 protein with the B55 class of regulatory subunits of protein phosphatase 2A (PP2A) and in yeast with the B55 homolog Cdc55. We have found previously that E4orf4 inhibits PP2A activity against at least some substrates. In an attempt to understand the mechanism of this inhibition, we used a genetic approach to identify residues in the seven-bladed β-propeller proteins B55α and Cdc55 required for E4orf4 binding. In both cases, amino-terminal polypeptides composed only of blade 1 and at least part of blade 2 were found to bind E4orf4 and overexpression blocked E4orf4 toxicity in yeast. Furthermore, certain amino acid substitutions in blades 1 and 2 within full-length B55α and Cdc55 resulted in loss of E4orf4 binding. Recent mutational analysis has suggested that segments of blades 1 and 2 present on the top face of B55α form part of the "substrate-binding groove." Additionally, these segments are in close proximity to the catalytic C subunit of the PP2A holoenzyme. Thus, our results are consistent with the hypothesis that E4orf4 binding could affect the access of substrates, resulting in the failure to dephosphorylate some PP2A substrates.
Insights
Human adenovirus E4orf4 protein toxicity stems from binding to protein phosphatase 2A (PP2A) regulatory subunits. Specific residues in B55α and Cdc55 blades 1 and 2 are crucial for this interaction, impacting PP2A activity.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Human adenovirus E4orf4 protein exhibits toxicity in human tumor cells and yeast.
- This toxicity is largely mediated by interactions with the B55 regulatory subunit class of protein phosphatase 2A (PP2A) and its yeast homolog, Cdc55.
- E4orf4 has been previously shown to inhibit PP2A activity.
Purpose of the Study:
- To elucidate the mechanism by which E4orf4 inhibits PP2A activity.
- To identify specific residues and protein domains in B55α and Cdc55 essential for E4orf4 binding using a genetic approach.
Main Methods:
- Genetic screening to identify residues in B55α and Cdc55 required for E4orf4 interaction.
- Analysis of amino-terminal polypeptides and specific amino acid substitutions in full-length proteins.
- Investigating the effect of E4orf4 binding on PP2A substrate dephosphorylation.
Main Results:
- Amino-terminal fragments of B55α and Cdc55, containing blade 1 and part of blade 2, were found to bind E4orf4.
- Overexpression of these fragments mitigated E4orf4-induced toxicity in yeast.
- Specific mutations within blades 1 and 2 of B55α and Cdc55 abolished E4orf4 binding.
- These critical regions are implicated in PP2A substrate binding and proximity to the catalytic subunit.
Conclusions:
- E4orf4 binds to a specific region on the B55α and Cdc55 subunits, involving blades 1 and 2.
- This binding likely interferes with substrate access to the PP2A holoenzyme.
- The findings support a model where E4orf4 binding inhibits PP2A by obstructing substrate dephosphorylation, leading to cellular toxicity.
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