Phosphatase Type 2A-dependent and -independent Pathways for ATR Phosphorylation of Chk1

• Published in: The Journal of biological chemistry 2007-03, Vol.282 (10), p.7287-7298
• Main Authors: Li, Ge, Elder, Robert T., Qin, Kefeng, Park, Hyeon Ung, Liang, Dong, Zhao, Richard Y.
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins - metabolism; Checkpoint Kinase 1; G2 Phase; Gene Products, vpr - physiology; HeLa Cells; Histones - metabolism; Humans; Hydroxyurea - pharmacology; Phosphoprotein Phosphatases - physiology; Phosphorylation; Protein Kinases - metabolism; Protein Phosphatase 2; Protein-Serine-Threonine Kinases - metabolism; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M607951200

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-Ser345 phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser345 phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G2 arrest and Cdk1-Tyr15 phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic β-isoform PP2A(Cβ) and the A regulatory α-isoform PP2A(Aα) are involved in the G2 induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr15 and Chk1-Ser345. In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser345 phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated γH2AX-Ser139 phosphorylation and foci formation, but down-regulation of PP2A(Aα) or PP2A(Cβ) did not decrease γH2AX-Ser139 phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aα/Cβ)-mediated G2 arrest and Chk1-Ser345 phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cβ) or PP2A(Aα) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aα/Cβ) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.