Abstract
ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser(345) phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser(345) phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G(2) arrest and Cdk1-Tyr(15) phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic beta-isoform PP2A(Cbeta) and the A regulatory alpha-isoform PP2A(Aalpha) are involved in the G(2) induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr(15) and Chk1-Ser(345). In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser(345) phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated gammaH2AX-Ser(139) phosphorylation and foci formation, but down-regulation of PP2A(Aalpha) or PP2A(Cbeta) did not decrease gammaH2AX-Ser(139) phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aalpha/Cbeta)-mediated G(2) arrest and Chk1-Ser(345) phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cbeta) or PP2A(Aalpha) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aalpha/Cbeta) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.
Highlights
To ensure accurate transmission of genetic information, eukaryotic cells have developed an elaborate network of checkpoints to monitor the successful completion of every cell cycle step and to respond to cellular abnormalities such as DNA
Inhibition of phosphatase type 2A (PP2A) Enzymatic Activity Reduces Vpr-induced G2 Arrest—okadaic acid (OA), a specific and potent inhibitor of PP2A, was first used to determine whether PP2A is involved in Vpr-induced G2 arrest
Vpr-induced G2 accumulation was significantly reduced when the concentrations of OA reached 17.5 and 25 nM. Treatment with these higher concentrations of OA on cells without vpr expression caused small increases of cells in G2 to 12.5–21.3%, the percentage of the G2 cells decreased from 62.1 to 37.1 and 37.3% in the vpr-expressing cells treated with these higher concentrations of OA (Fig. 1B). These results suggest that the activity of PP2A is required for Vpr-induced G2 arrest
Summary
To ensure accurate transmission of genetic information, eukaryotic cells have developed an elaborate network of checkpoints to monitor the successful completion of every cell cycle step and to respond to cellular abnormalities such as DNA. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser345 phosphorylation. The role of PP2A(A␣/C) in Vpr-induced phosphorylation of Cdk1-Tyr15 was evaluated in HeLa cells with the specific siRNAs against the PP2A subunits.
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