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J. Biol. Chem., Vol. 282, Issue 10, 7287-7298, March 9, 2007

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

Ge Li, Robert T. Elder, Kefeng Qin, Hyeon Ung Park, Dong Liang, and Richard Y. Zhao^¶||1

From the Department of Pathology, ^¶Department of Microbiology-Immunology, ^||Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60614

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 -isoform PP2A(C) and the A regulatory -isoform PP2A(A) 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 H2AX-Ser¹³⁹ phosphorylation and foci formation, but down-regulation of PP2A(A) or PP2A(C) did not decrease H2AX-Ser¹³⁹ phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(A/C)-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(C) or PP2A(A) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(A/C) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.

Received for publication, August 18, 2006 , and in revised form, January 5, 2007.

^* This study was supported in part by National Institute of Health Grants AI40891 and GM63080 (to R. Y. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ To whom correspondence should be addressed: Dept. of Pathology, University of Maryland School of Medicine, 10 South Pine St., MSTF700A, Baltimore, MD 21201. Tel.: 410-706-6301; Fax: 410-706-6302; E-mail: rzhao{at}som.umaryland.edu.

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