The adenovirus E4orf4 protein induces G2/M arrest and cell death by blocking protein phosphatase 2A activity...

Area of Science:

• Oncology
• Virology
• Molecular Biology

Background:

• Human adenovirus E4orf4 protein exhibits toxicity towards human tumor cells.
• The interaction between E4orf4 and the B alpha subunit of protein phosphatase 2A (PP2A) is crucial for its cell-killing activity.
• The precise mechanism by which E4orf4 binding affects PP2A activity remains unclear.

Purpose of the Study:

• To investigate the interaction of E4orf4 with PP2A B55 family subunits.
• To elucidate the effect of E4orf4 binding on B55-specific PP2A activity and its substrates.
• To determine the role of B55-specific PP2A inhibition in E4orf4-induced tumor cell toxicity and cell cycle arrest.

Main Methods:

• Assessed E4orf4 interaction with PP2A B55 family subunits.
• Measured in vitro B55-specific PP2A activity in the presence of E4orf4.
• Analyzed in vivo phosphorylation status of PP2A substrates (4E-BP1 and p70(S6K)) upon E4orf4 expression.
• Utilized phosphatase inhibitors (okadaic acid) and PP2A inhibitors (I(1)(PP2A)).
• Employed RNA interference targeting the B alpha subunit in H1299 cells.

Main Results:

• E4orf4 protein uniquely interacts with B55 family subunits, and cell killing correlates with E4orf4 expression levels.
• E4orf4 binding reduces B55-specific PP2A activity in vitro.
• Expression of E4orf4 leads to hypophosphorylation of 4E-BP1 and p70(S6K) in vivo.
• Inhibitors of PP2A activity (okadaic acid, I(1)(PP2A)) and B alpha-specific RNA interference enhance E4orf4-induced cell killing and G(2)/M arrest.

Conclusions:

• E4orf4 toxicity stems from the inhibition of B55-specific PP2A holoenzymes.
• E4orf4 induces cell growth arrest and death by reducing global B55-specific PP2A activity.
• This reduction in activity prevents the dephosphorylation of critical cell cycle progression substrates.