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J. Virol. doi:10.1128/JVI.01098-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

HIV-1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25

Department of Pathology, Department of Microbiology and Immunology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA, Children's Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA

^* To whom correspondence should be addressed. Email: rzhao{at}som.umaryland.edu.

	Abstract

HIV-1 Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G2 arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G2/M transition. Goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm, and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, as amino acids substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G2 arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G2 delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were co-expressed, implicating Srk1 and Vpr are likely affecting the cell cycle G2/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by siRNA or a MK2 inhibitor suppresses Vpr-induced cell cycle G2 arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G2 arrest at least in part through a Srk1/MK2-mediated mechanism.