Phosphatase type 2A-dependent and -independent pathways for ATR phosphorylation of Chk1.
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.
Li
Ge
G
Department of Pathology, Department of Microbiology-Immunology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Elder
Robert T
RT
Qin
Kefeng
K
Park
Hyeon Ung
HU
Liang
Dong
D
Zhao
Richard Y
RY
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D016195:G2 Phase; D016336:Gene Products, vpr / Q000502:physiology; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014466:Ultraviolet Rays
D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D018797:Cell Cycle Proteins; D016336:Gene Products, vpr; C468783:H2AX protein, human; D006657:Histones; C517191:PPP2R1A protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C517189:PPP2CA protein, human; C517190:PPP2CB protein, human; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D006918:Hydroxyurea