Advertisement
Protein Synthesis, Post-Translational Modification, and Degradation| Volume 283, ISSUE 7, P3846-3853, February 15, 2008

Download started.

Ok

Neuralized-like 1 (Neurl1) Targeted to the Plasma Membrane by N-Myristoylation Regulates the Notch Ligand Jagged1*

  • Evangelia Koutelou
    Footnotes
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110

    Joint Graduate Program in Molecular Biology and Biomedicine, Department of Biology, University of Crete, Heraklion, 71409, Crete, Greece
    Search for articles by this author
  • Shigeo Sato
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110
    Search for articles by this author
  • Chieri Tomomori-Sato
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110
    Search for articles by this author
  • Laurence Florens
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110
    Search for articles by this author
  • Selene K. Swanson
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110
    Search for articles by this author
  • Michael P. Washburn
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110
    Search for articles by this author
  • Maria Kokkinaki
    Footnotes
    Affiliations
    Department of Biology, University of Crete, Heraklion, 71409, Crete, Greece
    Search for articles by this author
  • Ronald C. Conaway
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110

    Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160
    Search for articles by this author
  • Joan W. Conaway
    Correspondence
    To whom correspondence may be addressed: Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110. Tel.: 816-926-4091; Fax: 816-926-2091
    Affiliations
    Stowers Institute for Medical Research, Kansas City, Missouri 64110

    Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160
    Search for articles by this author
  • Nicholas K. Moschonas
    Correspondence
    To whom correspondence may be addressed: Laboratory of Biology, School of Medicine, University of Patras, University Campus 26500, Patras, Greece. Tel.: 302610997689; Fax: 302610991769
    Affiliations
    Department of Biology, University of Crete, Heraklion, 71409, Crete, Greece
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Stowers Institute for Medical Research and by a Research and Education Action Program PYTHAGORAS II no. 2077 of the Greek Ministry of Education and the European Union (25% from national funds and 75% from the European Social Fund) (to N. K. M). 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) containssupplemental Table S1–S4, Figs. S1–S5, and other materials.
    1 Recipient of a predoctoral fellowship from the Propondis Foundation.
    2 Present address: Dept. of Biochemistry and Cell Biology, Georgetown University, Washington D. C. 20057.
Open AccessPublished:December 12, 2007DOI:https://doi.org/10.1074/jbc.M706974200
      Notch signaling constitutes an evolutionarily conserved mechanism that mediates cell-cell interactions in various developmental processes. Numerous regulatory proteins interact with the Notch receptor and its ligands and control signaling at multiple levels. Ubiquitination and endocytosis followed by endosomal sorting of both the receptor and its ligands is essential for Notch-mediated signaling. The E3 ubiquitin ligases, Neuralized (Neur) and Mind Bomb (Mib1), are crucial for regulating the activity and stability of Notch ligands in Drosophila; however, biochemical evidence that the Notch ligands are directly targeted for ubiquitination by Neur and/or Mib1 has been lacking. In this report, we explore the function of Neurl1, a mouse ortholog of Drosophila Neur. We show that Neurl1 can function as an E3 ubiquitin ligase to activate monoubiquitination in vitro of Jagged1, but not other mammalian Notch ligands. Neurl1 expression decreases Jagged1 levels in cells and blocks signaling from Jagged1-expressing cells to neighboring Notch-expressing cells. We demonstrate that Neurl1 is myristoylated at its N terminus, and that myristoylation of Neurl1 targets it to the plasma membrane. Point mutations abolishing either Neurl1 myristoylation and plasma membrane localization or Neurl1 ubiquitin ligase activity impair its ability to down-regulate Jagged1 expression and to block signaling. Taken together, our results argue that Neurl1 at the plasma membrane can affect the signaling activity of Jagged1 by directly enhancing its ubiquitination and subsequent turnover.
      Cells have evolved a complex machinery of signaling pathways to achieve coordination and fine programming of events that specify different cell types. Notch signaling plays an important role in cellular differentiation, proliferation, and apoptotic events at all stages of development (
      • Bray S.J.
      ). Notch proteins are members of a family of evolutionarily conserved transmembrane receptors that bind ligands of the Delta/Serrate/Lag1 (DSL)
      The abbreviations used are: DSL
      Delta/Serrate/Lag1
      NHR
      neuralized homology repeat
      Neurl1
      neuralized-like 1
      HA
      hemagglutinin
      GFP
      green fluorescent protein
      GST
      glutathione S-transferase.
      5The abbreviations used are: DSL
      Delta/Serrate/Lag1
      NHR
      neuralized homology repeat
      Neurl1
      neuralized-like 1
      HA
      hemagglutinin
      GFP
      green fluorescent protein
      GST
      glutathione S-transferase.
      type presented on juxtaposed cells and specify cell fate in a wide variety of tissues through local cell-cell interactions (
      • Artavanis-Tsakonas S.
      • Rand M.D.
      • Lake R.J.
      ). Genetic studies in Drosophila and Caenorhabditis elegans indicate that small differences in Notch receptor or ligand expression in adjacent cells are sufficient to alter Notch signaling, resulting in changes in cell fate decisions (
      • Greenwald I.
      ,
      • Heitzler P.
      • Simpson P.
      ). Nevertheless, activation of the Notch pathway can also be fine-tuned by additional mechanisms, including post-translational modifications and control of intracellular localization, because dramatic differences in signaling and signal reception between cells do not always correlate with obvious differences in the expression levels of ligands or receptors (
      • Schweisguth F.
      ).
      Recent advances have demonstrated essential roles for ubiquitination and endocytosis of receptors and ligands in Notch-mediated signaling (
      • Le Borgne R.
      • Bardin A.
      • Schweisguth F.
      ). Key components for the endocytosis of Notch ligands have been identified in Drosophila, zebrafish, and mice. Among these are proteins with general roles in the endocytic pathway, including liquid facets/epsin and auxilin, as well as RING finger ubiquitin ligases Mind bomb (Mib)-1, Mib-2, and Neuralized (Neur) (
      • Chen W.
      • Corliss D.C.
      ,
      • Hagedorn E.J.
      • Bayraktar J.L.
      • Kandachar V.R.
      • Bai T.
      • Englert D.M.
      • Chang H.C.
      ,
      • Itoh M.
      • Kim C.-H.
      • Palardy G.
      • Oda T.
      • Jiang Y.-J.
      • Maust D.
      • Yeo S.-Y.
      • Lorick K.
      • Wright G.J.
      • Ariza-McNaughton L.
      • Weissman A.M.
      • Lewis J.
      • Chandrasekharappa S.C.
      • Chitnis A.B.
      ,
      • Koo B.-H.
      • Lim H.-S.
      • Song R.
      • Yoon M.-J.
      • Yoon K.-J.
      • Moon J.-S.
      • Kim Y.-W.
      • Kwon M.-C.
      • Yoo K.-W.
      • Kong M.-P.
      • Lee J.
      • Chitnis A.B.
      • Kim C.-H.
      • Kong Y.-Y.
      ,
      • Koo B.-H.
      • Yoon K.-J.
      • Yoo K.-W.
      • Lim H.-S.
      • Song R.
      • Yoon M.-J.
      • Moon J.-S.
      • Kim Y.-W.
      • So J.-H.
      • Kim C.-H.
      • Kong Y.-Y.
      ,
      • Lai E.C.
      • Roegiers F.
      • Qin X.
      • Jan Y.N.
      • Rubin G.
      ,
      • Le Borgne R.
      • Remaud S.
      • Hamel S.
      • Schweisguth F.
      ,
      • Overstreet E.
      • Fitch E.
      • Fischer J.A.
      ,
      • Pitsouli C.
      • Delidakis C.
      ,
      • Wang W.
      • Struhl G.
      ,
      • Wang W.
      • Struhl G.
      ). In Drosophila, Neur and Mib regulate the Notch ligands Delta and Serrate by inducing their internalization and eventual degradation (
      • Itoh M.
      • Kim C.-H.
      • Palardy G.
      • Oda T.
      • Jiang Y.-J.
      • Maust D.
      • Yeo S.-Y.
      • Lorick K.
      • Wright G.J.
      • Ariza-McNaughton L.
      • Weissman A.M.
      • Lewis J.
      • Chandrasekharappa S.C.
      • Chitnis A.B.
      ,
      • Koo B.-H.
      • Lim H.-S.
      • Song R.
      • Yoon M.-J.
      • Yoon K.-J.
      • Moon J.-S.
      • Kim Y.-W.
      • Kwon M.-C.
      • Yoo K.-W.
      • Kong M.-P.
      • Lee J.
      • Chitnis A.B.
      • Kim C.-H.
      • Kong Y.-Y.
      ,
      • Lai E.C.
      • Roegiers F.
      • Qin X.
      • Jan Y.N.
      • Rubin G.
      ,
      • Le Borgne R.
      • Remaud S.
      • Hamel S.
      • Schweisguth F.
      ,
      • Pitsouli C.
      • Delidakis C.
      ,
      • Wang W.
      • Struhl G.
      ,
      • Lai E.C.
      • Deblandre G.A.
      • Kintner C.
      • Rubin G.M.
      ,
      • Pavlopoulos E.
      • Pitsouli C.
      • Klueg K.M.
      • Muskavitch M.A.T.
      • Moschonas N.K.
      • Delidakis C.
      ). Ubiquitination of the ligands seems to be necessary not only for their turnover, but also for downstream trafficking events that render the ligands active for signaling; however, the nature of these downstream events remains unclear (
      • Le Borgne R.
      • Bardin A.
      • Schweisguth F.
      ).
      In mice, there are two Neuralized-like genes, Neurl1 and Neurl2 (
      • Pavlopoulos E.
      • Kokkinaki M.
      • Koutelou E.
      • Mitsiadis T.A.
      • Prinos P.
      • Delidakis C.
      • Kilpatrick M.W.
      • Tsipouras P.
      • Moschonas N.K.
      ,
      • Ruan Y.
      • Tecott L.
      • Jiang M.-M.
      • Jan L.Y.
      • Jan Y.N.
      ,
      • Song R.
      • Koo B.-K.
      • Yoon K.-J.
      • Yoon M.-J.
      • Yoo K.-W.
      • Kim H.-T.
      • Oh H.-J.
      • Kim Y.-Y.
      • Han J.-K.
      • Kim C.-H.
      • Kong Y.-Y.
      ,
      • Vollrath B.
      • Pudney J.
      • Asa S.
      • Leder P.
      • Fitzgerald K.
      ). Like Drosophila Neur, they encode proteins that consist of a C-terminal RING finger domain and two neuralized homology repeat (NHR) domains, NHR1 and NHR2. Recently, Neurl2 has been shown to exhibit a nonspecific ubiquitin ligase activity in vitro. Coexpression of Neurl2 with mammalian Delta-like 1 (Dll1) was shown to promote increased Dll1 ubiquitination in cells. In addition, Neurl2 functioned cooperatively with Mib1 to promote endocytosis of XDelta to Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate)-positive early endosomes (
      • Song R.
      • Koo B.-K.
      • Yoon K.-J.
      • Yoon M.-J.
      • Yoo K.-W.
      • Kim H.-T.
      • Oh H.-J.
      • Kim Y.-Y.
      • Han J.-K.
      • Kim C.-H.
      • Kong Y.-Y.
      ); however, unequivocal evidence that Dll1 is a direct target of Neurl2 ubiquitin ligase activity is lacking. Although Neurl1 has also been shown to exhibit a nonspecific ubiquitin ligase activity in vitro (
      • Song R.
      • Koo B.-K.
      • Yoon K.-J.
      • Yoon M.-J.
      • Yoo K.-W.
      • Kim H.-T.
      • Oh H.-J.
      • Kim Y.-Y.
      • Han J.-K.
      • Kim C.-H.
      • Kong Y.-Y.
      ), putative Neurl1 substrates have not been identified. In addition, whether Neurl1 plays a role in Notch signaling is unclear, because Neurl1–/– mice exhibit no abnormal cell fate specifications during neurogenesis and somitogenesis, two processes in which Notch signaling has been shown to be involved (
      • Ruan Y.
      • Tecott L.
      • Jiang M.-M.
      • Jan L.Y.
      • Jan Y.N.
      ,
      • Vollrath B.
      • Pudney J.
      • Asa S.
      • Leder P.
      • Fitzgerald K.
      ).
      In this report, we demonstrate that Neurl1 is a RING-dependent E3 ubiquitin ligase that can regulate Notch signaling in mammals. Neurl1 specifically targets the intracellular domain of Jagged1, but not of other Notch ligands, for monoubiquitination in vitro. Further, we show that Neurl1 expression promotes lysosomal degradation of Jagged1 in cells and blocks signaling from Jagged1-expressing cells to neighboring Notch-expressing cells. Finally, we present evidence that Neurl-dependent turnover of Jagged1 and inhibition of Notch signaling depends upon N-terminal myristoylation and plasma membrane localization of Neurl1.

      EXPERIMENTAL PROCEDURES

      Plasmid Construction–The Neurl1 cDNA was obtained from the I.M.A.G.E. Consortium (MGC:66919 IMAGE: 6831952). The Neurl1 open reading frame was amplified by PCR and introduced into pCR 2.1-TOPO Plasmids expressing Flag-tagged full-length or truncated forms of Neurl1 were amplified via PCR from pCR2.1-TOPO containing Neurl1 and subcloned with an N- or C-terminal Flag tag into pcDNA3.1 (Invitrogen) or into pBacPAK8 (Clontech) (seesupplemental Table S1 for more details). Plasmids encoding Neurl1-Rm-F and Neurl1G2A-F were generated from pcDNA3.1 Neurl1-F using site-directed mutagenesis as described (
      • Ho S.N.
      • Hunt H.D.
      • Horton R.M.
      • Pullen J.K.
      • Pease L.R.
      ). pBOS-SN3T, a mammalian expression vector encoding HA-tagged Jagged1 ligand (pBOS-SN3T, (
      • Lindsell C.E.
      • Shawber C.J.
      • Boulter J.
      • Weinmaster G.
      ) was provided by G. Weinmaster (University of California, Los Angeles, CA) and p3xFlagDIP1(Mib1) plasmid (
      • Jin Y.
      • Blue E.K.
      • Dixon S.
      • Shao Z.
      • Gallagher P.J.
      ) was provided by P. J. Gallagher (Department of Cellular and Integrative Physiology, Indianapolis, IN). Plasmid pIRES J1-HA was generated by introducing J1-HA cDNA in XbaI restriction site of pIRES vector (Clontech) and Neurl1-FpIRESJ1-HA plasmid was generated by introducing Neurl1-Flag in NheI and XhoI restriction sites of pIRES vector.
      Cell Lines–Parental cell lines were obtained from the American Type Culture Collection and propagated as recommended. To generate clonal L cell lines stably expressing C-terminally HA-tagged Jagged1 (J1-HA) or C-terminally Flag-tagged Neurl1 and C-terminally HA-tagged Jagged1 (J1-HA-Neurl1-F), plasmids pIRES-J1HA or Neurl1-F-pIRES-J1HA were introduced into cells by electroporation, and G-418 resistant clones were isolated. Jagged1-HA or Neurl1-Flag expression was confirmed by immunoprecipitation with agarose beads conjugated to anti-HA (Sigma A2220) or anti-Flag (Sigma A2095) antibodies, followed by immunoblotting.
      Transfection, Immunoblotting, and Immunoprecipitation–Human embryonic kidney (HEK) 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics. Cells were transfected with appropriate amounts of plasmid DNA using Fugene 6 (Roche Applied Sciences). At 36-h post-transfection, cells were harvested in 0.5 ml of lysis buffer (50 mm Tris, pH 7.9, 150 mm NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate) containing protease inhibitors (Calbiochem, 539131). Protein samples were separated by SDS/PAGE and transferred to Protran Nitrocellulose membranes (Whatman) and visualized by immunoblotting with either Supersignal West Dura or Supersignal West Femto chemiluminescent reagents (Pierce). HEK293T cells were transiently transfected with the indicated plasmids and harvested 36 h after transfection in buffer containing 50 mm HEPES-NaOH, pH 7.9, 5 mm MgCl2, 150 mm NaCl, 0.2% Triton X-100, 20% glycerol and 1× protease inhibitor mixture (Calbiochem 539131). Lysates were incubated for 4–5 h with 50 μl of anti-Flag (M2)-agarose beads equilibrated in the same buffer. The beads were washed three times with 10 mm HEPES/NaOH (pH 7.9), 1.5 mm MgCl2, 150 mm NaCl, 10 mm KCl, and 0.2% Triton X-100, and bound proteins were eluted with 10 mm HEPES/NaOH (pH 7.9), 1.5 mm MgCl2, 100 mm NaCl, and 0.05% Triton X-100 containing 0.2 mg/ml Flag peptide (Sigma, F3290).
      Immunofluorescence–HEK293T cells were grown on coverslips, fixed with 2% paraformaldehyde, washed in phosphate-buffered saline, blocked with 5% bovine serum albumin in 0.2% Triton and phosphate-buffered saline (PBS-T) and stained using mouse anti-Flag M2 antibody (Sigma, F3165) and Alexa 488-conjugated anti-mouse antibody (Invitrogen). Images were obtained using a laser scanning spectral confocal microscope (Leica TCS SP2).
      Expression and Purification of Recombinant Proteins in Sf21 Insect Cells–Recombinant baculoviruses were generated with the BacPAK baculovirus expression system (Clontech). Sf21 cells were cultured at 27 °C in Sf-900 II SFM with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). Sixty hours after infection with appropriate recombinant baculoviruses, Sf21 cells were collected in ice-cold buffer containing 50 mm Hepes-NaOH (pH 7.9), 150 mm NaCl, 5 mm MgCl2, 0.2% Triton X-100, 20% glycerol, and protease inhibitors (Calbiochem, 539131). After centrifugation at 100,000 × g for 1 h at 4 °C, the resulting supernatant was mixed with 0.2 ml of anti-Flag (M2)-agarose or anti-HA agarose beads for 4–5 h. The beads were washed three times with washing buffer (10 mm HEPES/NaOH (pH 7.9), 1.5 mm MgCl2, 150 mm NaCl, 10 mm KCl, and 0.2% Triton X-100) and bound proteins were eluted from the beads with elution buffer (10 mm HEPES/NaOH (pH 7.9), 1.5 mm MgCl2, 100 mm NaCl, and 0.05% Triton X-100) containing 0.2 mg/ml Flag peptide or 0.4 mg/ml HA peptide.
      In Vitro Ubiquitination Assays–Wild type or mutant Neurl1, expressed in and purified from Sf21 cells, was incubated for 1 h at 37 °C with ≈50 nm Uba1, ≈0.2 μm UbcH5a, UbcH2, UbcH3, UbcH6 or UbcH7, and 0.25 mg/ml ubiquitin, with or without 2 μg of JAG1ICD-HA in 20-μl reactions containing 50 mm Tris-HCl, pH 7.6, 50 mm NaCl, 5 mm MgCl2, 0.5 mm EDTA (pH 7.9), 5% glycerol, 2 mm ATP, 0.5 mm dithiothreitol, 0.3 units/ml pyrophosphatase, 60 mm creatine phosphate, and 0.3 mg/ml creatine phosphokinase. N-terminally (His)6-tagged mouse E1 was expressed in Sf21 insect cells and purified by nickel-agarose chromatography as described (
      • Iwai K.
      • Yamanaka K.
      • Kamura T.
      • Minato N.
      • Conaway R.C.
      • Conaway J.W.
      • Klausner R.D.
      • Pause A.
      ). Human UbcH5a with an N-terminal 6-histidine tag and a C-terminal Flag tag were expressed in E. coli strain BL21(DE3) and purified by nickel agarose (
      • Iwai K.
      • Yamanaka K.
      • Kamura T.
      • Minato N.
      • Conaway R.C.
      • Conaway J.W.
      • Klausner R.D.
      • Pause A.
      ). Recombinant human UbcH3, UbcH6, and UbcH7 were purchased from Sigma (U2383, U9007, and U9132, respectively), and GST-tagged human UbcH2 was purchased from Boston Biochem (E2–605).
      In Vivo Ubiquitination Assays–HEK293T cells were transiently transfected in 150-mm dishes with plasmids encoding His-Ub and either F-Mib1 or Neurl1-F. Cells were lysed in 800 μl/dish of 6 m guanidium-HCl, 100 mm Na2HPO4, 10 mm imidazole, and protease inhibitors (Calbiochem, 539131). Lysates were cleared by ultracentrifugation (100,000 × g for 1 h at 4 °C). His-Ub-conjugated proteins were purified by nickel chromatography using Ni-NTA-agarose (Qiagen). Nickel-binding proteins were analyzed by immunoblotting using anti-Flag antibody.
      Coculture Assay–C33A cells were plated at 70% confluency. The next day, parental L cells or cells stably expressing either J1-HA or J1-HA and Neurl1-F were added at a density equal to the monolayer of C33A cells. RNA was extracted after 0, 8, or 12 h of co-culture. When HEK293T cells were used in the coculture assay instead of L cells, they were transiently transfected with the indicated plasmids and plated on C33A monolayers 36-h post-transfection. RT-PCR was performed using 50–100 ng of RNA with the ImProm II Reverse Transcription system (Promega) according to the manufacturer's protocol, using the following primers: HES1 forward, 5′-GATGCTCTGAAGAAAGATAGCTCGCGG-3′; HES1 reverse, 5′-CACCGCCCCCACCGGGACC-3′. Real-time PCR was performed using iQ SYBR Green SuperMix and the MyiQ Single-Color Real-Time PCR Detection System (BioRad) according to the manufacturer's protocol. All real-time PCR assays were performed in triplicate in at least three independent experiments. Notch-induced activation of HES1 is expressed as a ratio of HES1 RNA, normalized to actin and detected in ligand-expressing cells, compared with HES1 RNA, normalized to actin and detected in parental L cells.
      Identification of Neurl1 Myristoylation Site by Mass Spectrometry–C-terminally Flag-tagged Neurl1 was immuno-purified from stably transformed HEK293 cells and analyzed by MudPIT mass spectrometry (
      • Washburn M.P.
      • Wolters D.
      • Yates J.R.
      • II I
      ,
      • Wolters D.A.
      • Washburn M.P.
      • Yates J.R.
      • II I
      ) as described in supplemental methods andsupplemental Table S2 to identify peptides containing myristoylated glycines (+210).

      RESULTS

      Neurl1 Targets Jagged1 ICD for Ubiquitination in Vitro–In initial experiments, we found that Neurl1, expressed in and purified from baculovirus-infected Sf21 insect cells as a C-terminally Flag-tagged protein (Neurl-F, Fig. 1A), promotes formation of polyubiquitin chains when incubated with ubiquitin, ATP, the E1 ubiquitin-activating enzyme Uba1, and the E2 ubiquitin-conjugating enzyme UbcH5a (supplemental Fig. S1 and Fig. 1B). To determine whether the activity of Neurl1 is RING-dependent we employed a truncated form of Neurl1 lacking the RING domain (NHR1 + 2-F) and a Neurl1 mutant in which two highly conserved RING domain cysteine residues, Cys521 and Cys524, have been changed to serine (Neurl1Rm-F, Fig. 1A). As expected, neither NHR1 + 2-F nor Neurl1Rm-F supported formation of polyubiquitin chains in vitro (Fig. 1B, lanes 4 and 5). In contrast, a Neurl1 mutant, F-NHR2+R, which has an intact RING domain but lacks NHR1, was fully active (Fig. 1B, lane 6), consistent with previous reports that an intact RING domain is usually sufficient for nonspecific ubiquitin ligase activity (
      • Lorick K.L.
      • Jensen J.P.
      • Fang S.
      • Ong A.M.
      • Hatakeyama S.
      • Weissman A.M.
      ). In addition, we observed a shift in the electrophoretic mobility of Neurl1-F itself, particularly when reactions were performed at high E2 concentrations (Fig. 1B, lanes 3, 9, and 10), suggesting that Neurl1 can be autoubiquitinated.
      Figure thumbnail gr1
      FIGURE 1Neurl1 is a RING-dependent ubiquitin ligase that supports ubiquitination of the Jagged1 ICD in vitro. A, schematic diagram of Neurl1 proteins used in this study. B, Neurl1 supports E1 and UbcH5a-dependent formation of polyubiquitin chains. 1 μg of the indicated Neurl1 protein was assayed for nonspecific ubiquitin ligase activity as described under “Experimental Procedures” with either 0.2 μm (lanes 2–8) or .0.2, 0.1, or 0.05 μm (lanes 9–11) UbcH5a. C, indicated Neurl1 proteins were assayed for the ability to support ubiquitination of Jagged1 ICD. Reactions included either 1 μg of Neurl1-Flag (lanes 1–2, 4–6, 8) or 0.5 μg of Neurl1-Flag (lane 7) with E1 and UbcH5a as indicated. Reaction products were detected by immunoblotting with either anti-HA or anti-Flag antibodies. The intense smear detected with anti-Flag in lane 6 is due to multi-ubiquitin chains containing Flag-ubiquitin. The asterisk denotes a nonspecific band detected in anti-Flag immunoblots. Ubn, polyubiquitin; F-Ub, Flag-ubiquitin; IB, immunoblot; J1ICD, Jagged1 ICD.
      To gain insight into the potential role of Neurl1 in ubiquitination of Notch ligands, we used a biochemical approach to investigate whether any of the ligands can be directly targeted by mammalian Neurl1. Incubation of recombinant Jagged1 intracellular domain (ICD), expressed in and purified from baculovirus-infected Sf21 cells (Fig. 1C, lanes 7 and 8), but not the ICDs of Dll1, Dll4, or Jagged2 (supplemental Fig. S2), with recombinant ubiquitin, E1, E2, and Neurl1-F led to the appearance of a more slowly migrating species of the size expected of monoubiquitinated Jagged1 ICD. Confirming that this species was indeed monoubiquitinated, we observed that it migrated still more slowly when we replaced ubiquitin with Flag epitope-tagged ubiquitin, which has a slightly larger molecular mass than untagged ubiquitin (Fig. 1C, lane 6). As expected, monoubiquitination of Jagged1 ICD depends on the integrity of the Neurl1 RING domain, because Neurl1Rm failed to support ICD ubiquitination (Fig. 1C, lane 5). Although it appears from our biochemical assays that Neurl1 ubiquitin ligase activity is specific for Jagged1, it is important to note that substrate recognition by many ubiquitin ligases depends upon appropriate post-translational modification of the substrate (
      • Deshaies R.
      ). Hence, we cannot rule out the possibility that Neurl1 fails to support ubiquitination of other DSL ligands in our assays because they are not suitably modified when expressed in insect cells.
      The Neurl1 NHR2 Domain Mediates Interaction with Jagged1Drosophila and Xenopus Neuralized have been reported to interact with Delta through the Neur NHR1 domain (
      • Commisso C.
      • Boulianne G.L.
      ). To define the region of Neurl1 needed for interaction with Jagged1, we co-expressed Flag-tagged Neurl1 or Neurl1 mutants with HA-tagged Jagged1 ICD in Sf21 cells (Fig. 2, A and B) and with full-length HA-tagged Jagged1 in HEK293 cells (Fig. 2C). As expected, we were able to detect stable binding of Neurl1-F and HA-tagged Jagged or Jagged1 ICD in coimmunoprecipitations from HEK293 or insect cell lysates, respectively. The RING domain was dispensable for these interactions. In lysates of baculovirus-infected insect cells, the NHR1 domain interacted weakly with the Jagged1 ICD (Fig. 2B, lane 5); however the interaction was much more robust when NHR2 was present (Fig. 2B, lane 6). Similarly, a small amount of full-length Jagged1 coimmunoprecipitated with NHR1 from HEK293T cells, while a more robust interaction was detected when both NHR domains or NHR2 alone was present (Fig. 2C, lanes 5 and 8). Taken together, these results suggest that NHR2 is primarily responsible for the interaction of Neurl1 with the Jagged1 ICD.
      Figure thumbnail gr2
      FIGURE 2Neurl1 binds the Jagged1 ICD. Lysates of Sf21 cells infected with baculoviruses encoding wild type Neurl1 (A) or the indicated Neurl1 proteins (B) were immunoprecipitated with the indicated antibodies and detected by immunoblotting. C, lysates of HEK293T cells transfected with pcDNA3.1 or pcDNA3.1 encoding the indicated proteins was subjected to immunoprecipitation and immunoblotting.
      Neurl1 Is N-terminally Myristoylated and Targeted to the Plasma MembraneIn silico analysis of the mouse Neurl1 N-terminal sequence predicted that Neurl1 might be myristoylated at its second glycine residue (Fig. 3A). MS/MS analysis confirmed that this residue is indeed myristoylated in Neurl1 expressed in cells (Fig. 3B andsupplemental Table S2). Peptides containing unmodified G2 were never detected, suggesting that all or most Neurl1 is myristoylated. Whether other Neuralized proteins are myristoylated remains to be determined; however, in silico analyses predict with high confidence that one of the two isoforms of Drosophila Neuralized, but not mouse Neurl2, has a myristoylation site, while Xenopus Neurl has a possible myristoylation site (supplemental Table S3).
      Figure thumbnail gr3
      FIGURE 3Neurl1 is myristoylated and targeted to the plasma membrane. A, schematic drawing of Neurl1 mutant constructs. B, annotated tandem mass spectrum matched to M.myrGNNFSSVSSLQR.G from mouse Neurl1. The doubly charged precursor ion with a protonated mass of 1508.69 Da is marked by the green tick under the m/z-axis, while b and y ion series are labeled in red and blue, respectively. Water neutral losses are denoted with *. C, HEK293T cells were transiently transfected with plasmids encoding Neurl1-Flag or Neurl1G2A-Flag. Flag epitope was detected with mouse anti-Flag antibody, followed by Alexa 488-conjugated anti-mouse antibody (green). Nuclei were counterstained with DAPI (blue), and cells were analyzed by confocal microscopy using a 40× objective. D and E, HEK293T cells were transiently transfected with plasmids encoding the indicated proteins, and cell lysates were immunoblotted with anti-Flag and anti-β-tubulin antibodies.
      Although Drosophila Neur has been localized to the plasma membrane, Neurl1 has been reported to have a largely cytoplasmic or nuclear localization based on analyses of Neurl1 fused at its N terminus to EGFP or other tags (
      • Pavlopoulos E.
      • Kokkinaki M.
      • Koutelou E.
      • Mitsiadis T.A.
      • Prinos P.
      • Delidakis C.
      • Kilpatrick M.W.
      • Tsipouras P.
      • Moschonas N.K.
      ,
      • Ruan Y.
      • Tecott L.
      • Jiang M.-M.
      • Jan L.Y.
      • Jan Y.N.
      ,
      • Vollrath B.
      • Pudney J.
      • Asa S.
      • Leder P.
      • Fitzgerald K.
      ), which would be predicted to interfere with myristoylation. To begin to explore the functional significance of Neurl1 myristoylation, we compared the subcellular localization of C-terminally Flag-tagged Neurl1 and a Neurl1 mutant in which the second glycine was changed to alanine (Neurl1G2A). By confocal microscopy of transfected cells, we observed that a substantial fraction of wild-type Neurl1 was localized to the plasma membrane in 94 ± 2% of transfected cells. In contrast, Neurl1G2A was localized to the plasma membrane in only 4 ± 2% of transfected cells and instead was detected primarily in vesicular structures in the cytoplasm (Fig. 3C and supplemental Fig. S4, andsupplemental Table S4).
      Mutation of the myristoylation site affected not only Neurl1 localization, but also levels of Neurl1 protein in cells. Drosophila and Xenopus Neuralized are expressed at very low levels due to rapid ubiquitin-dependent degradation by the proteasome (
      • Lai E.C.
      • Deblandre G.A.
      • Kintner C.
      • Rubin G.M.
      ,
      • Talora Claudio
      • Sgroi Dennis C.
      • Crum Christopher P.
      • Dotto a.G. P.
      ). Similarly, we observed that wild-type Neurl1 is ubiquitinated and expressed at very low levels when introduced by transient transfection into HEK293T cells, but that it is stabilized by treatment of cells with the proteasome inhibitor MG132 (supplemental Fig. S3). Deletion or mutation of the RING domain had a modest effect on Neurl1 expression (Fig. 3, D and E). Neurl1G2A, as well as Neurl1 mutants lacking the N-terminal NHR1 domain, accumulated to substantially higher levels (Fig. 3, D and E), suggesting that Neurl1 localized to the plasma membrane by myristoylation is destabilized.
      Neurl1 Down-regulates Jagged1 Expression–Our observations that Neurl1 interacts with Jagged1 and supports monoubiquitination of the Jagged1 ICD in vitro suggested that Neurl1 might ubiquitinate Jagged1 and regulate its stability in cells. Consistent with that possibility, we observe that expression of Jagged1 with wild-type Neurl1, but not Neurl1 with a mutated RING domain, leads to decreased accumulation of Jagged1 protein in cells. In the experiments of Fig. 4, A and B, HEK293 T cells were transfected with a plasmid encoding HA-tagged Jagged1, with or without plasmids encoding Neurl1 or Neurl1 mutants, and the level of Jagged1 expression was determined by Western blotting with anti-HA antibodies at various times after transfection. Thirty-six hours after transfection, Jagged1 was readily detectable whether or not Neurl1 was present (Fig. 4A). In the absence of Neurl1, Jagged1 levels remained relatively constant throughout the time course of the experiment, whereas in cells expressing Neurl1, Jagged1 was dramatically reduced by 48-h post-transfection (Fig. 4A). Jagged1 levels remained constant in the presence of Neurl1Rm, suggesting that loss of Jagged1 depends on Neurl1 ubiquitin ligase activity (Fig. 4B). In addition, although Neurl1G2A can interact with Jagged1 (Fig. 4C), Jagged1 levels remained constant in the presence of Neurl1G2A (Fig. 4B). Thus, Neurl1-dependent regulation of Jagged1 depends on Neurl1 myristoylation and/or plasma membrane localization.
      Figure thumbnail gr4
      FIGURE 4Neurl1-induced down-regulation of Jagged1. A, HEK293T cells were transiently transfected with plasmids encoding the indicated proteins. At various times after transfection, cells were harvested, and cell lysates were analyzed either by direct immunoblotting with anti-HA or anti-β-tubulin antibodies or by immunoprecipitation by anti-Flag antibodies followed by immunoblotting with anti-Flag. B, HEK293T cells were transiently transfected with plasmids encoding the indicated proteins. At various times after transfection, expression of Neurl1-F and Jagged1-HA was measured as in A. C, HEK293T cells were transiently transfected with empty vector or plasmids encoding and the indicated proteins. 36 h after transfection, cells were harvested and analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. D, HEK293T cells were transiently transfected with plasmids encoding 6-histidine-tagged ubiquitin or ubiquitin K0, Jagged1-HA, and either Neurl1-F or Neurl1Rm-F. 36 h after transfection, cultures were supplemented with 50 μm MG132 and grown for an additional 12 h. Upper panel, cells were lysed in buffer containing 6 m guanidine HCl, and histidine-tagged, ubiquitinated proteins were affinity-purified on nickel-agarose under denaturing conditions. Eluates from nickel-agarose chromatography were analyzed by HA immunoblotting. Middle panel, cells were lysed under non-denaturing conditions, and Jagged1-HA was immunoprecipitated from cell lysates with anti-HA antibody and detected by immunoblotting with anti-HA. Lower panel, cells were lysed under non-denaturing conditions, and Neurl1-Flag or Neurl1Rm-Flag was immunoprecipitated from cell lysates with anti-Flag antibody and detected by immunoblotting with anti-Flag. E, assay the indicated proteins for their effects on Notch signaling, coculture assays using transiently transfected HEK293T cells as the signaling cells were performed as described under “Experimental Procedures.”
      To determine whether Neurl1 expression affects the ubiquitination status of Jagged1 in cells, HA-tagged Jagged1 was expressed with or without 6-histidine-tagged ubiquitin and Flag-tagged Neurl1 or the inactive Neurl1 RING mutant. Cell lysates were then subjected to nickel agarose chromatography under denaturing conditions to purify proteins that were covalently conjugated to ubiquitin. As shown in Fig. 4D, the amount of ubiquitinated Jagged1 recovered from nickel agarose was increased by coexpression of wild-type Neurl1, but not the Neurl1 RING mutant. The electrophoretic mobility of ubiquitinated Jagged1 from cells expressing 6-histidine-tagged ubiquitin and 6-histidine-tagged ubiquitin K0, which has no lysines and therefore cannot assemble into polyubiquitin chains, was indistinguishable, arguing that Neurl1 most likely supports monoubiquitination of Jagged1 in cells.
      Ubiquitination can serve as a signal directing proteins for degradation by the proteasome or, in the case of transmembrane proteins, for internalization and degradation by the lysosome (
      • Hicke L.
      ). We therefore examined the effects on Jagged1 expression in Neurl expressing cells of epoxomycin, a potent and highly specific proteasome inhibitor, MG132, a broader spectrum protease inhibitor that blocks proteasomal and, to a lesser extent, lysosomal proteases (
      • Lee D.H.
      • Goldberg A.L.
      ), and chloroquine, an inhibitor of lysosomal function (supplemental Fig. S4). Epoxomicin had little or no effect on Jagged1 expression, while MG132 treatment led to a modest increase. In contrast, Jagged1 expression remained relatively constant throughout the time course of the experiment in the presence of chloroquine, suggesting that down-regulation of Jagged1 by Neurl1 ubiquitin ligase is largely dependent on lysosomal protease activity.
      Neurl1 Overexpression Reduces Jagged1 Signaling Activity in Cells–Productive Notch signaling in cells leads to the activation of target genes like mammalian Hairy/Enhancer of Split (HES1), a basic helix-loop-helix protein that blocks cellular differentiation (
      • Iso T.
      • Kedes L.
      • Hamamori Y.
      ). To measure directly and quantitatively the effect of mouse Neurl1 on Notch signaling, HEK293T cells were transiently transfected with Jagged1 alone or Jagged1 and Neurl1. Transfected cells were cocultured with C33A cells, which endogenously express Notch1 and Notch2 (
      • Talora Claudio
      • Sgroi Dennis C.
      • Crum Christopher P.
      • Dotto a.G. P.
      ). Activation of the Notch signaling pathway results in translocation of Notch1 and Notch2 intracellular domain into the C33A cell nuclei and activation of the endogenous HES1 gene. HES1 gene transcription was then detected using quantitative real-time PCR. HES1 mRNA was strongly induced after coculture of C33A cells with cells expressing Jagged1 alone (Fig. 4D). Notch signaling was blocked by expression of Neurl1, because HES1 mRNA was not induced after coculture of C33A cells with cells cotransfected with both Jagged1 and Neurl1. Consistent with our observation that Jagged1 was expressed at high levels in the presence of the Neurl1 RING domain mutant or Neurl1G2A, Notch signaling was strongly induced in cells expressing either of these Neurl1 mutants.
      To corroborate the results obtained in assays using transiently transfected HEK293T cells, we measured Jagged1 expression and Jagged1-dependent activation of the HES1 gene in L cells stably expressing Jagged1 or both Jagged1 and Neurl1. As observed in transiently transfected cells, Neurl1 expression led to a dramatic decrease of Jagged1 expression in L cells (Fig. 5A); this decrease was most likely due to a change in Jagged1 stability, because Jagged1 expression could be partially restored by treatment of cells with the proteasome inhibitor MG132 (Fig. 5B). Consistent with the results obtained in transiently transfected cells, Jagged1-induced signaling was dramatically reduced in cells stably expressing Neuralized (Fig. 5C). Interestingly, signaling was partially restored by treatment of cells with MG132 (Fig. 5D).
      Figure thumbnail gr5
      FIGURE 5Neurl1 overexpression reduces Jagged1 signaling activity in cells. L cells stably expressing Jagged1-HA or Jagged1-HA and Neurl1-F were cultured in the absence of MG132 (A) or with MG132 for the indicated times (B). Cell lysates were analyzed either by direct immunoblotting with anti-HA or anti-β-tubulin antibodies or by immunoprecipitation by anti-Flag antibodies followed by immunoblotting with anti-Flag. Coculture assays using L cells stably expressing Jagged1-HA or Jagged1-HA and Neurl1-F as the signaling cells were performed as described under “Experimental Procedures,” with or without MG132 indicated in the figure (C and D).

      DISCUSSION

      Although mouse Neurl1 was first described over five years ago (
      • Ruan Y.
      • Tecott L.
      • Jiang M.-M.
      • Jan L.Y.
      • Jan Y.N.
      ,
      • Vollrath B.
      • Pudney J.
      • Asa S.
      • Leder P.
      • Fitzgerald K.
      ), its potential role in ubiquitination and regulation of Notch pathway components has not been evaluated. In Drosophila, it is well established that the ubiquitin ligase activities of Neuralized and/or Mindbomb1 are needed for endocytosis, activation, and degradation of Notch ligands, all processes likely dependent on ubiquitination of the ligand intracellular domains (
      • Lai E.C.
      • Roegiers F.
      • Qin X.
      • Jan Y.N.
      • Rubin G.
      ,
      • Le Borgne R.
      • Remaud S.
      • Hamel S.
      • Schweisguth F.
      ,
      • Pitsouli C.
      • Delidakis C.
      ,
      • Wang W.
      • Struhl G.
      ,
      • Glittenberg M.
      • Pitsouli C.
      • Garvey C.
      • Delidakis C.
      • Bray S.
      ). Furthermore, ectopic expression of a second murine Neur paralog, Neurl2, was shown to increase ubiquitination of Dll1 in transfected cells (
      • Song R.
      • Koo B.-K.
      • Yoon K.-J.
      • Yoon M.-J.
      • Yoo K.-W.
      • Kim H.-T.
      • Oh H.-J.
      • Kim Y.-Y.
      • Han J.-K.
      • Kim C.-H.
      • Kong Y.-Y.
      ), and a Xenopus Neurl ortholog was shown to promote ubiquitination of in vitro translated Xenopus Delta1 (
      • Deblandre G.A.
      • Lai E.C.
      • Kintner C.
      ). Nevertheless, until the present work it had not been rigorously demonstrated, using a purified enzyme system, that a Neuralized protein directly supports ubiquitination of a ligand. Here, we use an in vitro ubiquitination system, reconstituted with purified, recombinant proteins, to provide direct biochemical evidence that Neurl1 acts as a RING-dependent ubiquitin ligase that supports monoubiquitination of Jagged1. In addition, we show that overexpression of Neurl1 leads to RING-dependent down-regulation of Jagged1 in cells, consistent with the model that the ubiquitin ligase activity observed in vitro contributes to Neurl1 activity in cells. Both Drosophila and mammalian Neur and Mib have been shown to interact with various Notch ligands (
      • Koo B.-H.
      • Lim H.-S.
      • Song R.
      • Yoon M.-J.
      • Yoon K.-J.
      • Moon J.-S.
      • Kim Y.-W.
      • Kwon M.-C.
      • Yoo K.-W.
      • Kong M.-P.
      • Lee J.
      • Chitnis A.B.
      • Kim C.-H.
      • Kong Y.-Y.
      ,
      • Lai E.C.
      • Roegiers F.
      • Qin X.
      • Jan Y.N.
      • Rubin G.
      ,
      • Pitsouli C.
      • Delidakis C.
      ,
      • Lai E.C.
      • Deblandre G.A.
      • Kintner C.
      • Rubin G.M.
      ,
      • Song R.
      • Koo B.-K.
      • Yoon K.-J.
      • Yoon M.-J.
      • Yoo K.-W.
      • Kim H.-T.
      • Oh H.-J.
      • Kim Y.-Y.
      • Han J.-K.
      • Kim C.-H.
      • Kong Y.-Y.
      ). For Drosophila Neur and mouse Neurl2, this interaction has been mapped to one or both NHR domains (
      • Song R.
      • Koo B.-K.
      • Yoon K.-J.
      • Yoon M.-J.
      • Yoo K.-W.
      • Kim H.-T.
      • Oh H.-J.
      • Kim Y.-Y.
      • Han J.-K.
      • Kim C.-H.
      • Kong Y.-Y.
      ,
      • Talora Claudio
      • Sgroi Dennis C.
      • Crum Christopher P.
      • Dotto a.G. P.
      ). The Neurl1-Jagged1 interaction also appears to require the two NHR domains, particularly NHR2. Although we also observed interactions of Neurl1 with Delta1 and Jagged2 in mammalian cells, only Jagged1 was ubiquitinated in our reconstituted in vitro system, and only Jagged1 bound to Neurl1 when the proteins were coexpressed in insect cells (data not shown). We do not yet know what accounts for this difference; however, it is conceivable that the interaction of Neurl1 with Delta1 and Jagged2 depends upon additional proteins and/or on post-translational modifications that are present only in mammalian cells.
      The subcellular localization of Neurl1 has remained controversial. Consistent with its role as a regulator of Notch ligand ubiquitination and internalization, Drosophila neuralized has been shown to be a plasma membrane-associated protein (
      • Pavlopoulos E.
      • Pitsouli C.
      • Klueg K.M.
      • Muskavitch M.A.T.
      • Moschonas N.K.
      • Delidakis C.
      ). In contrast, Neurl1 has been reported to exhibit a punctate, largely cytoplasmic and perinuclear (
      • Pavlopoulos E.
      • Kokkinaki M.
      • Koutelou E.
      • Mitsiadis T.A.
      • Prinos P.
      • Delidakis C.
      • Kilpatrick M.W.
      • Tsipouras P.
      • Moschonas N.K.
      ,
      • Ruan Y.
      • Tecott L.
      • Jiang M.-M.
      • Jan L.Y.
      • Jan Y.N.
      ) or even a nuclear (
      • Timmusk T.
      • Palm K.
      • Belluardo N.
      • Mudo G.
      • Neuman T.
      ) localization based on analyses of N-terminal GFP fusion proteins.
      Our evidence that localization of Neurl1 to the plasma membrane depends upon its modification by N-terminal myristoylation provides an explanation for this apparent discrepancy. The addition of myristate and other fatty acids to the N termini of proteins facilitates reversible protein targeting to the plasma membrane (
      • Sigal C.T.
      • Zhou W.
      • Buser C.A.
      • McLaughlin S.
      • Resh M.D.
      ). Co-translational or post-translational N-myristoylation of proteins on an amino-terminal glycine is catalyzed by N-myristoyltransferases (NMTs) (
      • Farazi T.A.
      • Waksman G.
      • Gordon J.I.
      ,
      • Towler D.A.
      • Gordon J.I.
      ) and only occurs subsequent to removal of the initiating methionine by methionine aminopeptidase. Importantly, methionine aminopeptidase cannot cut after an internal methionine; hence, fusion of Neurl1 to an N-terminal epitope tag or GFP will block N-myristoylation. Consistent with the possibility that the failure in previous studies to observe localization of Neurl1 to the plasma membrane was due to the presence of the N-terminal GFP, we find that while C-terminally Flag-tagged Neurl1 localizes to the plasma membrane, N-terminally Flag-tagged Neurl1, like the nonmyristoylateable Neurl1G2A mutant, exhibits punctate cytoplasmic staining by immunofluorescence (data not shown).
      Myristoylation-dependent membrane localization of Neurl1 appears important for Neurl1 ability to interact functionally with Jagged1. Because the Neurl1G2A mutant can be immunoprecipitated from cell lysates with Jagged1, myristoylation is not essential for physical interaction of Neurl1 with Jagged1. Nevertheless, Neurl1G2A cannot down-regulate Jagged1 expression in cells, nor can it block its signaling activity in coculture assays, an observation that could suggest that Jagged1 and Neurl must be independently targeted to the plasma membrane before they can functionally interact with one another in intact cells. Alternatively, down-regulation of Jagged1 expression and activity could require that Neurl1 is targeted to specific plasma membrane microdomains by myristoylation. In this regard, it is noteworthy that some proteins, among which are certain Src family kinases and the HIV-1 pathogenicity factor Nef, can be targeted to lipid rafts by N-myristoylation (
      • Mukherjee A.
      • Arnaud L.
      • Cooper J.A.
      ,
      • Resh M.D.
      ,
      • Giese S.I.
      • Woerz I.
      • Homann S.
      • Tibroni N.
      • Geyer M.
      • Fackler O.T.
      ).
      Our observation that Neurl1 expression attenuates, rather than potentiates, Jagged1 activity is somewhat surprising in light of prior evidence from Drosophila that Neurl functions as a positive regulator of Notch signaling. The partial recovery of signaling activity upon inhibition of Jagged1 degradation after treatment of cells with MG132 suggests that the loss of ability to signal to Notch is due to ligand degradation. In other systems, ligand degradation has been shown to occur concurrently with signaling activation (
      • Le Borgne R.
      • Remaud S.
      • Hamel S.
      • Schweisguth F.
      ,
      • Wang W.
      • Struhl G.
      ,
      • Pavlopoulos E.
      • Pitsouli C.
      • Klueg K.M.
      • Muskavitch M.A.T.
      • Moschonas N.K.
      • Delidakis C.
      ). It is not clear whether the same ubiquitination event(s) that are responsible for ligand activation also mediate targeting to a degradation pathway. It is possible that there are two types of ubiquitin ligases for DSL proteins, some of which, like Neur PA, the best studied isoform of Drosophila Neuralized, favor activation (
      • Lai E.C.
      • Deblandre G.A.
      • Kintner C.
      • Rubin G.M.
      ,
      • Pavlopoulos E.
      • Pitsouli C.
      • Klueg K.M.
      • Muskavitch M.A.T.
      • Moschonas N.K.
      • Delidakis C.
      ,
      • Deshaies R.
      ), while others, such as mouse Neurl1 down-regulate the ligand, leading to inactivation. It is noteworthy that the Xenopus Neuralized ortholog has also been reported to antagonize Notch signaling by down-regulating levels of membrane-associated Delta1 (
      • Deblandre G.A.
      • Lai E.C.
      • Kintner C.
      ). Alternatively, it is possible that all Neuralized proteins can either activate or inactivate DSL ligands, depending on context. Consistent with this possibility, in Drosophila ectopic expression assays, high neuralized overexpression biases toward Notch inactivation, whereas lower overexpression biases toward Notch activation (
      • Lai E.C.
      • Rubin G.M.
      ). Whether the observed down-regulation of Jagged1 by Neurl1 reflects its normal function or whether it is also capable of activating Notch signaling when expressed at sufficiently low levels or in different cellular contexts remains to be determined.

      Acknowledgments

      We thank Tingting Yao and Elias Pavlopoulos for useful comments, and Gerry Weinmaster and Patricia Gallagher for Jagged1 and Mindbomb1 expression plasmids. We are particularly grateful to Christos Delidakis for helpful suggestions and critical reading of the manuscript.

      Supplementary Material

      References

        • Bray S.J.
        Nat. Rev. Mol. Cell Biol. 2006; 7: 678-689
        • Artavanis-Tsakonas S.
        • Rand M.D.
        • Lake R.J.
        Science. 1999; 284: 770-776
        • Greenwald I.
        Genes Dev. 1998; 12: 1751-1762
        • Heitzler P.
        • Simpson P.
        Cell. 1991; 64: 1083-1092
        • Schweisguth F.
        Curr. Biol. 2004; 14: R129-R138
        • Le Borgne R.
        • Bardin A.
        • Schweisguth F.
        Development. 2005; 132: 1751-1762
        • Chen W.
        • Corliss D.C.
        Dev. Biol. 2004; 267: 361-373
        • Hagedorn E.J.
        • Bayraktar J.L.
        • Kandachar V.R.
        • Bai T.
        • Englert D.M.
        • Chang H.C.
        J. Cell Biol. 2006; 173: 443-452
        • Itoh M.
        • Kim C.-H.
        • Palardy G.
        • Oda T.
        • Jiang Y.-J.
        • Maust D.
        • Yeo S.-Y.
        • Lorick K.
        • Wright G.J.
        • Ariza-McNaughton L.
        • Weissman A.M.
        • Lewis J.
        • Chandrasekharappa S.C.
        • Chitnis A.B.
        Dev. Cell. 2003; 4: 67-82
        • Koo B.-H.
        • Lim H.-S.
        • Song R.
        • Yoon M.-J.
        • Yoon K.-J.
        • Moon J.-S.
        • Kim Y.-W.
        • Kwon M.-C.
        • Yoo K.-W.
        • Kong M.-P.
        • Lee J.
        • Chitnis A.B.
        • Kim C.-H.
        • Kong Y.-Y.
        Development. 2005; 132: 3459-3470
        • Koo B.-H.
        • Yoon K.-J.
        • Yoo K.-W.
        • Lim H.-S.
        • Song R.
        • Yoon M.-J.
        • Moon J.-S.
        • Kim Y.-W.
        • So J.-H.
        • Kim C.-H.
        • Kong Y.-Y.
        J. Biol. Chem. 2005; 280: 22335-22342
        • Lai E.C.
        • Roegiers F.
        • Qin X.
        • Jan Y.N.
        • Rubin G.
        Development. 2005; 132: 2319-2332
        • Le Borgne R.
        • Remaud S.
        • Hamel S.
        • Schweisguth F.
        PLoS. 2005; 3: e96
        • Overstreet E.
        • Fitch E.
        • Fischer J.A.
        Development. 2004; 131: 5355-5366
        • Pitsouli C.
        • Delidakis C.
        Development. 2005; 132: 4041-4050
        • Wang W.
        • Struhl G.
        Development. 2004; 131: 5367-5380
        • Wang W.
        • Struhl G.
        Development. 2005; 132: 2883-2894
        • Lai E.C.
        • Deblandre G.A.
        • Kintner C.
        • Rubin G.M.
        Dev. Cell. 2001; 1: 783-794
        • Pavlopoulos E.
        • Pitsouli C.
        • Klueg K.M.
        • Muskavitch M.A.T.
        • Moschonas N.K.
        • Delidakis C.
        Dev. Cell. 2001; 1: 1-10
        • Pavlopoulos E.
        • Kokkinaki M.
        • Koutelou E.
        • Mitsiadis T.A.
        • Prinos P.
        • Delidakis C.
        • Kilpatrick M.W.
        • Tsipouras P.
        • Moschonas N.K.
        Biochim. Biophys. Acta. 2002; 1574: 375-382
        • Ruan Y.
        • Tecott L.
        • Jiang M.-M.
        • Jan L.Y.
        • Jan Y.N.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9907-9912
        • Song R.
        • Koo B.-K.
        • Yoon K.-J.
        • Yoon M.-J.
        • Yoo K.-W.
        • Kim H.-T.
        • Oh H.-J.
        • Kim Y.-Y.
        • Han J.-K.
        • Kim C.-H.
        • Kong Y.-Y.
        J. Biol. Chem. 2006; 281: 36391-36400
        • Vollrath B.
        • Pudney J.
        • Asa S.
        • Leder P.
        • Fitzgerald K.
        Mol. Cell Biol. 2001; 21: 7481-7494
        • Ho S.N.
        • Hunt H.D.
        • Horton R.M.
        • Pullen J.K.
        • Pease L.R.
        Gene. 1989; 77: 51-59
        • Lindsell C.E.
        • Shawber C.J.
        • Boulter J.
        • Weinmaster G.
        Cell. 1995; 80: 908-917
        • Jin Y.
        • Blue E.K.
        • Dixon S.
        • Shao Z.
        • Gallagher P.J.
        J. Biol. Chem. 2002; 277: 46980-46986
        • Iwai K.
        • Yamanaka K.
        • Kamura T.
        • Minato N.
        • Conaway R.C.
        • Conaway J.W.
        • Klausner R.D.
        • Pause A.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12436-12441
        • Washburn M.P.
        • Wolters D.
        • Yates J.R.
        • II I
        Nat. Biotech. 2001; 19: 242-247
        • Wolters D.A.
        • Washburn M.P.
        • Yates J.R.
        • II I
        Anal. Chem. 2001; 73: 5683-5690
        • Lorick K.L.
        • Jensen J.P.
        • Fang S.
        • Ong A.M.
        • Hatakeyama S.
        • Weissman A.M.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369
        • Deshaies R.
        Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467
        • Commisso C.
        • Boulianne G.L.
        Mol. Biol. Cell. 2007; 18: 1-13
        • Hicke L.
        Nat. Rev. Mol. Cell Biol. 2001; 2: 195-201
        • Lee D.H.
        • Goldberg A.L.
        Trends Cell Biol. 1998; 8: 397-403
        • Iso T.
        • Kedes L.
        • Hamamori Y.
        J. Cell Phys. 2003; 194: 237-255
        • Talora Claudio
        • Sgroi Dennis C.
        • Crum Christopher P.
        • Dotto a.G. P.
        Genes Dev. 2002; 16: 2252-2263
        • Glittenberg M.
        • Pitsouli C.
        • Garvey C.
        • Delidakis C.
        • Bray S.
        EMBO J. 2006; : 1-10
        • Deblandre G.A.
        • Lai E.C.
        • Kintner C.
        Dev. Cell. 2001; 1: 795-806
        • Timmusk T.
        • Palm K.
        • Belluardo N.
        • Mudo G.
        • Neuman T.
        Mol. Cell Neurosci. 2002; 20: 649-668
        • Sigal C.T.
        • Zhou W.
        • Buser C.A.
        • McLaughlin S.
        • Resh M.D.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12253-12257
        • Farazi T.A.
        • Waksman G.
        • Gordon J.I.
        J. Biol. Chem. 2001; 276: 39501-39504
        • Towler D.A.
        • Gordon J.I.
        Annu. Rev. Biochem. 1988; 57: 69-97
        • Mukherjee A.
        • Arnaud L.
        • Cooper J.A.
        J. Biol. Chem. 2003; 278: 40806-40814
        • Resh M.D.
        Nat. Chem. Biol. 2006; 2: 584-590
        • Giese S.I.
        • Woerz I.
        • Homann S.
        • Tibroni N.
        • Geyer M.
        • Fackler O.T.
        Virology. 2006; 355: 175-191
        • Lai E.C.
        • Rubin G.M.
        Dev. Biol. 2001; 231: 217-233