The Rockefeller University Press $30.00 J. Cell Biol. Vol. 196 No. 1 29–36 www.jcb.org/cgi/doi/10.1083/jcb.201105009 JCB 29 JCB: Report J. Howitt, J. Lackovic, and L.-H. Low contributed equally to this paper. Correspondence to Lloyd C. Trotman: trotman@cshl.edu; or Seong-Seng Tan: stan@ florey.edu.au Abbreviations used in this paper: CCA, common carotid artery; HI, hypoxia- ischemia; KO, knockout; MEF, mouse embryonic fibroblast; pAkt, phosphorylated Akt; RCCAo, right carotid artery occlusion; WT, wild type. Introduction PTEN (phosphatase and tensin homologue deleted on chromo- some TEN) is the major negative regulator of signaling by phos- phatidylinositol 3-kinase (PI 3-K), thereby playing a central role in controlling many important cellular activities regulated by this pathway, including cell division, cell growth, cell survival, and DNA damage (Chalhoub and Baker, 2009). PTEN exerts its negative effect through its phosphatase activity on the plasma membrane lipid phosphatidylinositol 3,4,5-triphosphate (PIP3), reducing levels of phosphorylated Akt (pAkt; Maehama and Dixon, 1998; Stambolic et al., 1998). Thus, loss of PTEN, as demonstrated by genetic inactivation in human cancer or mouse knockout (KO) models, causes constitutive activation of Akt in cells, resulting in dysregulated cell proliferation, growth, and sur- vival, which are hallmarks of tumorigenesis (Hobert and Eng, 2009; Nardella et al., 2010). PTEN can be found in both the cyto- plasm and nucleus of many cell and tissue types, and its aberrant localization has been implicated in disease. The nucleocytoplas- mic distribution of PTEN has been proposed to affect its tumor- suppressive function both within and outside the PI 3-K pathway (Planchon et al., 2008). However, it has remained unclear what physiological stimulus can drive PTEN into the nucleus and under what in vivo circumstances this can occur. In the brain, PTEN is required for multiple aspects of neuronal function and development, including maintenance of neuron structure, size, synaptic plasticity, and survival (Endersby P TEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regula- tor of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiologi- cal events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was trans- located into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family–interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overex- pression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1 / fibro- blasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival. Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia Jason Howitt, 1 Jenny Lackovic, 1 Ley-Hian Low, 1 Adam Naguib, 4 Alison Macintyre, 1 Choo-Peng Goh, 1 Jennifer K. Callaway, 2 Vicki Hammond, 1 Tim Thomas, 3 Matthew Dixon, 3 Ulrich Putz, 1 John Silke, 3 Perry Bartlett, 5 Baoli Yang, 6 Sharad Kumar, 7 Lloyd C. Trotman, 4 and Seong-Seng Tan 1 1 Brain Development and Regeneration Laboratory, Florey Neuroscience Institutes, 2 Department of Pharmacology, and 3 Walter and Eliza Hall Institute of Medical Research, The University of Melbourne, Parkville, Victoria 3010, Australia 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 5 Queensland Brain Institute, University of Queensland, St. Lucia QLD 4072, Australia 6 Department of Obstetrics and Gynecology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242 7 Center for Cancer Biology, SA Pathology, Adelaide SA 5000, Australia © 2012 Howitt et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). THE JOURNAL OF CELL BIOLOGY on August 5, 2014 jcb.rupress.org Downloaded from Published January 2, 2012 http://jcb.rupress.org/content/suppl/2011/12/29/jcb.201105009.DC1.html Supplemental Material can be found at: