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The Ubiquitin Ligase Nedd4-1 Is Dispensable for the Regulation of PTEN Stability and Localization

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The Ubiquitin Ligase Nedd4-1 Is Dispensable for the Regulation of PTEN Stability and Localization The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization Fatemeh Fouladkou†‡, Tamara Landry‡§, Hiroshi Kawabe¶, Antje Neeb¶, Chen Lu†, Nils Brose¶, Vuk Stambolic§ʈ, and Daniela Rotin†ʈ †Hospital for Sick Children and Biochemistry Department, University of Toronto, MaRS-TMDT, 101 College Street, Toronto, Ontario M5G 1L7, Canada; §Ontario Cancer Institute, University Health Network, 610 University Avenue, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada; and ¶Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, Go¨ttingen 37075, Germany Communicated by Aaron J. Ciechanover, Technion-Israel Institute of Technology, Bat Galim, Haifa, Israel, April 4, 2008 (received for review December 5, 2007) PTEN is a tumor suppressor frequently mutated in cancer. Recent location of this tumor suppressor to the nucleus, where it was reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that protected from degradation (22). regulates its stability and nuclear localization. We tested the Nedd4-1 is an E3 ubiquitin ligase that contains a C2 domain, physiological role of Nedd4-1 as a PTEN regulator by using cells and three or four WW domains, and a ubiquitin ligase Hect domain, and tissues derived from two independently generated strains of mice it belongs to the Nedd4 family of Hect E3 ligases that also includes with their Nedd4-1 gene disrupted. PTEN stability and ubiquitina- its relative Nedd4-2 (23–25). The WW domain of Nedd4 proteins tion were indistinguishable between the wild-type and Nedd4-1- recognizes and binds a short amino acid sequence in substrate deficient cells, and an interaction between the two proteins could proteins, called the PY motif (L/PPxY) (26–30). By using two not be detected. Moreover, PTEN subcellular distribution, showing independent approaches, we recently generated distinct strains of prominent cytoplasmic and nuclear staining, was independent of mice with disrupted Nedd4-1 gene, leading to a complete loss of Nedd4-1 presence. Finally, activation of PKB/Akt, a major down- Nedd4-1 protein and embryonic lethality at mid to late gestation stream target of cytoplasmic PTEN activity, and the ability of PTEN (F.F. and D.R., unpublished observations; H.K., A.N., and N.B., to transactivate the Rad51 promoter, a measure of its nuclear unpublished observations). Surprisingly, by using a comprehensive function, were unaffected by the loss of Nedd4-1. Taken together, analysis of PTEN levels, subcellular distribution, and cytoplasmic BIOCHEMISTRY our results fail to support a role for Nedd4-1 as the E3 ligase and nuclear function in tissues and cells from Nedd4-1 knockout regulating PTEN stability and subcellular localization. mice, we demonstrate here that Nedd4-1 is dispensable for regu- lation of PTEN stability, subcellular localization, and activity. E3 ligase ͉ tumor suppressor ͉ P13K signal ͉ WW domain Results TEN is one of the most frequently mutated genes in human Analysis of PTEN Levels and Ubiquitination in Nedd4-1-Deficient Cells. Pcancer (1–3). In the cytoplasm, PTEN functions as a lipid To examine the physiological function of Nedd4-1, its gene in the phosphatase by dephosphorylating the D3 position of phosphati- mouse was disrupted by using two independent approaches. First, ␤ dylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], and directly antago- by using gene-trap methodology, a -gal cDNA was inserted into nizing PI3K (4–6). Consistent with a negative regulatory role for the mouse Nedd4-1 genomic locus between exons 6 and 7 (ES trap PTEN in regulation of PI3 signaling, PTEN-deficient cells and clone XB786; BayGenomics) (F.F. and D.R., unpublished obser- tissues exhibit defects in cell proliferation, growth, survival, death, vations). In a separate effort, exons 9 and 10 of mouse Nedd4-1 protein translation, metabolism, migration, and structural organi- (Ensemble NC࿝000075.4) were disrupted by homologous recombi- zation (2, 3, 7, 8). nation in ES cells (H.K., A.N., and N.B., manuscript in prepara- Ϫ Ϫ One of the most intriguing features of PTEN is its subcellular tion). Nedd4-1 / embryos resulting from both knockout strategies localization. Although PTEN was originally found to be a die at mid to late gestation displaying complete loss of Nedd4-1 cytoplasmic protein (9), its nuclear localization in many cell types protein (F.F. and D.R., unpublished observations; and H.K., A.N., has been reported by using various independently developed and N.B., unpublished observations). To explore the physiological monoclonal and polyclonal anti-PTEN antibodies (10–14). A function of Nedd4-1 in greater detail, day 13.5–14.5 mouse embry- Ϫ Ϫ number of attempts have been made to uncouple the cytoplasmic onic fibroblasts (MEFs) from Nedd4-1 / embryos, as well as from and nuclear roles of PTEN, yielding incongruous results. For their sibling wild-type (WT) and heterozygote controls, were example, differing accounts of the respective levels of cytoplas- generated by using two different strategies. Serial passage was used Ϫ Ϫ mic and nuclear PTEN throughout the stages of the cell cycle to derive the Nedd4-1 / trap MEFs (31), whereas MEFs from Ϫ Ϫ have been documented (15, 16). Functionally, nuclear PTEN has Nedd4-1 / exon9,10 were immortalized by retrovirally mediated ex- been shown to induce cell cycle arrest in certain cell types (17), pression of the SV40 Large-T antigen. Immunoblotting of protein whereas in others, nuclear accumulation of PTEN increased lysates from these cells demonstrated complete loss of Nedd4-1 upon stimulation with proapoptotic factors and correlated with expression in Nedd4-1Ϫ/Ϫtrap and Nedd4-1Ϫ/Ϫexons9,10 cells, whereas induction of apoptosis (18). Finally, nuclear-targeted PTEN was shown to impede the growth of U251MG glioblastoma cells and down-regulate p70S6K in a PKB/Akt-independent manner but Author contributions: F.F., T.L., V.S., and D.R. designed research; F.F., T.L., and H.K. performed research; H.K., A.N., C.L., N.B., V.S., and D.R. contributed new reagents/analytic was unable to inhibit cell invasion (19). tools; F.F., T.L., C.L., V.S., and D.R. analyzed data; and F.F., T.L., V.S., and D.R. wrote the Recently, published work revealed a phosphatase activity- paper. independent nuclear role for PTEN in the regulation of chromo- The authors declare no conflict of interest. somal stability and repair of DNA damage (20). Wang et al. (21) and ‡F.F. and T.L. contributed equally to this work. Trotman et al. (22) reported that PTEN stability and nuclear ʈTo whom correspondence may be addressed. E-mail: [email protected] or vuks@ translocation were regulated by ubiquitination mediated by the uhnres.utoronto.ca. ubiquitin ligase Nedd4-1. Although the Nedd4-1-mediated polyu- This article contains supporting information online at www.pnas.org/cgi/content/full/ biquitination of PTEN in the cytosol caused PTEN degradation 0803233105/DCSupplemental. (21), its Nedd4-1-dependent monoubiquitination led to the trans- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803233105 PNAS ͉ June 24, 2008 ͉ vol. 105 ͉ no. 25 ͉ 8585–8590 Downloaded by guest on October 2, 2021 MEFs: +/+ +/- -/- MEFs: +/+ +/- -/- ________MEFs ____________________ Hearts A bp B A kDa +/+ -/- +/+ +/- -/- +/+ -/- IB: kDa 120- Nedd4-1 anti IB: 64- PTEN 84- 850- Nedd4-1 anti PTEN 650- 67- _ 500 _ 400 IB: actin IB: anti actin actin 49- anti actin 120- Hearts: +/+ +/- +/- -/- IB: Nedd4-2 84- IB: anti anti Nedd4-2 Nedd4-1 67- Nedd4-1 IB: actin IB: anti actin actin anti actin µg protein: 10 10 10 10 40 40 µg protein: 10 10 10 10 40 40 MEFs: +/+ -/- +/+ -/- +/+ -/- C MEFs +/+ -/- +/+ -/- +/+ -/- IB: 64- anti PTEN IB: PTEN Nedd4-1 anti Nedd4-1 IB: anti actin actin ________ ___________________ IB: anti actin actin -/-,trap -/-, exon9,10 MEFs: Nedd4-1 ________ ___________________ Nedd4-1 -/-trap -/-exon9,10 MEF line: Nedd4-1 Nedd4-1 ____________Nedd4-1 RNAi B HEK293T Fig. 1. Loss of Nedd4-1 expression in MEFs derived from Nedd4-1 knock- _ ϩ/ϩ transfection: non-silencingshRNA(1)shRNA(2)shRNA(3) out mice. (A) Genotyping of MEFs generated from Nedd4-1 , Nedd4- kDa ϩ Ϫ Ϫ Ϫ IB: anti 1 / trap, and Nedd4-1 / trap embryos. PCR analysis of genomic DNA from 115- Nedd4-1 Nedd4-1 the indicated cells is shown. A 447-bp fragment corresponds to the WT allele and a 680-bp fragment to the trapped allele. (B) Immunoblotting of IB: anti - 64- PTEN lysates from Nedd4-1ϩ/ϩ, Nedd4-1ϩ/Ϫtrap, and Nedd4-1Ϫ/Ϫtrap MEFs. Cell PTEN IB: anti- lysates were probed for Nedd4-1 and Nedd4–2 by using anti-Nedd4-1 actin actin antibodies (Upper) and anti-Nedd4–2 antibodies (Lower), respectively. Actin levels were determined with anti-actin antibodies. (C ) Immunoblot- -1) ϩ ϩ Ϫ Ϫ ting of lysates from Nedd4-1 / and Nedd4-1 / exons9,10 MEFs. Ten or 40 ␮g C MEFs(wt) _ RNA(Nedd4 of proteins obtained from cell lysates of the indicated MEFs were probed transfection: non-silencingsh for Nedd4-1 by immunoblotting with anti-Nedd4-1 antibodies, as in B. IB: anti Nedd4-1115- Nedd4-1 Actin levels were determined with anti-actin antibodies. IB: anti PTEN 64- PTEN actin the expression of its close relative Nedd4–2 remained unaffected (Fig. 1). D MEFs(-/-) 1 _ _ Nedd4- By using in vitro ubiquitination assays and monitoring PTEN transfection: GFP-Nedd4-1 GFP- ubiquitination upon overexpression or knockdown of Nedd4-1 in 180- IB: anti GFP cultured cells, a direct role of Nedd4-1, but not Nedd4-2, in 115- GFP-Nedd4-1 regulation of PTEN ubiquitination and stability was recently pro- IB: anti PTEN 64- PTEN posed (21).
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