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• W2502332015 abstract "Article25 July 2016free access Source DataTransparent process Synaptonuclear messenger PRR7 inhibits c-Jun ubiquitination and regulates NMDA-mediated excitotoxicity Dana O Kravchick Dana O Kravchick Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Anna Karpova Anna Karpova Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Matous Hrdinka Matous Hrdinka Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Jeffrey Lopez-Rojas Jeffrey Lopez-Rojas Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Sanda Iacobas Sanda Iacobas Department of Pathology, New York Medical College, Valhalla, NY, USA Search for more papers by this author Abigail U Carbonell Abigail U Carbonell Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Dumitru A Iacobas Dumitru A Iacobas Department of Pathology, New York Medical College, Valhalla, NY, USA Search for more papers by this author Michael R Kreutz Michael R Kreutz Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Leibniz Group “Dendritic Organelles and Synaptic Function”, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Bryen A Jordan Corresponding Author Bryen A Jordan orcid.org/0000-0002-4967-444X Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Dana O Kravchick Dana O Kravchick Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Anna Karpova Anna Karpova Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Matous Hrdinka Matous Hrdinka Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Jeffrey Lopez-Rojas Jeffrey Lopez-Rojas Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Search for more papers by this author Sanda Iacobas Sanda Iacobas Department of Pathology, New York Medical College, Valhalla, NY, USA Search for more papers by this author Abigail U Carbonell Abigail U Carbonell Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Dumitru A Iacobas Dumitru A Iacobas Department of Pathology, New York Medical College, Valhalla, NY, USA Search for more papers by this author Michael R Kreutz Michael R Kreutz Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany Leibniz Group “Dendritic Organelles and Synaptic Function”, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Bryen A Jordan Corresponding Author Bryen A Jordan orcid.org/0000-0002-4967-444X Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Author Information Dana O Kravchick1, Anna Karpova2, Matous Hrdinka2, Jeffrey Lopez-Rojas2, Sanda Iacobas3, Abigail U Carbonell1, Dumitru A Iacobas3, Michael R Kreutz2,4 and Bryen A Jordan 1,5 1Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA 2Research Group Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germany 3Department of Pathology, New York Medical College, Valhalla, NY, USA 4Leibniz Group “Dendritic Organelles and Synaptic Function”, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, NY, USA *Corresponding author. Tel: +1 718 430 2675; E-mail: [email protected] The EMBO Journal (2016)35:1923-1934https://doi.org/10.15252/embj.201593070 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Elevated c-Jun levels result in apoptosis and are evident in neurodegenerative disorders such as Alzheimer's disease and dementia and after global cerebral insults including stroke and epilepsy. NMDA receptor (NMDAR) antagonists block c-Jun upregulation and prevent neuronal cell death following excitotoxic insults. However, the molecular mechanisms regulating c-Jun abundance in neurons are poorly understood. Here, we show that the synaptic component Proline rich 7 (PRR7) accumulates in the nucleus of hippocampal neurons following NMDAR activity. We find that PRR7 inhibits the ubiquitination of c-Jun by E3 ligase SCFFBW7 (FBW7), increases c-Jun-dependent transcriptional activity, and promotes neuronal death. Microarray assays show that PRR7 abundance is directly correlated with transcripts associated with cellular viability. Moreover, PRR7 knockdown attenuates NMDAR-mediated excitotoxicity in neuronal cultures in a c-Jun-dependent manner. Our results show that PRR7 links NMDAR activity to c-Jun function and provide new insights into the molecular processes that underlie NMDAR-dependent excitotoxicity. Synopsis The transcription factor c-Jun regulates NMDA receptor-dependent neuronal death. Proline rich 7 (PRR7) is a synaptic protein that shuttles to the nucleus upon NMDA receptor stimulation, upregulates c-Jun abundance by inhibiting its ubiquitination, and plays a role in neuronal excitotoxicity. PRR7 binds to NMDA receptors and is a novel activity-dependent synaptonuclear messenger in hippocampal neurons. PRR7 inhibits the ubiquitination of c-Jun by the SCF-complex E3 ligase, FBW7. PRR7 nuclear transport elevates c-Jun abundance, increases c-Jun transcriptional activity, and promotes neuronal death. PRR7 activity regulates transcripts associated with cellular viability, and loss of PRR7 is neuroprotective. Introduction The transcription factor c-Jun is a principal component of the activator protein 1 (AP-1) complex, which is involved in the immediate early response following neuronal activity (Raivich & Behrens, 2006). To form the AP-1 complex, c-Jun can homo- or heterodimerize with members of the Fos or ATF families of transcription factors. AP-1 is required for neuronal survival and synaptic plasticity, and has been implicated in seizures, addiction, pain, and posttraumatic repair in the CNS (Alberini, 2009). Despite considerable interest in AP-1 biology, relatively little is known about how neuronal activity regulates c-Jun protein levels (Cruzalegui et al, 1999; Tischmeyer & Grimm, 1999). NMDAR activity increases c-Jun abundance in several neurodegenerative disorders and following ischemia or status epilepticus (Dragunow et al, 1993; Herdegen et al, 1997; Zhang et al, 2006). In the retina, excitotoxic NMDAR stimulation upregulates c-Jun mRNA and protein abundance (Munemasa et al, 2006) and triggers c-Jun phosphorylation at amino acid residues 63/73 by activating c-Jun N-terminal kinases (JNK) (Yang et al, 1997; Coffey, 2014). Phosphorylated c-Jun is transcriptionally active and can induce apoptosis via upregulation of cell death-inducing genes (Ham et al, 1995; Behrens et al, 1999; Song et al, 2011) or by downregulating anti-apoptotic genes through repressor activity (Shaulian & Karin, 2002; Miao & Ding, 2003). Although numerous studies implicate c-Jun in apoptosis, its cellular function is context and stimulus dependent (Leppa & Bohmann, 1999) as c-Jun can also regulate axon regeneration and synaptic plasticity (Alberini, 2009; Hu et al, 2015). In neurons, c-Jun protein levels are kept low by rapid proteasomal-mediated degradation to prevent cellular apoptosis (Nateri et al, 2004). The HECT-type E3-ligase Itch (Fang et al, 2002), MEKK1 (Xia et al, 2007), and RING type E3 ligase SCFFBW7 complex (composed of Skp1, Cullin1, and the F-box protein FBW7) (Nateri et al, 2004) can ubiquitinate and promote c-Jun proteasomal degradation. Ubiquitination by FBW7 requires c-Jun phosphorylation at Ser63/73 and Thr91/93. NMDAR stimulation increases c-Jun phosphorylation and transcriptional activity by activating JNK (Schwarzschild et al, 1997). While MAPK-dependent phosphorylation can stabilize c-Jun expression in 3T3 cells (Musti et al, 1997), factors that protect c-Jun from polyubiquitination and degradation in neurons are unknown. Several groups, including our own, identified PRR7 in mass spectrometry-based screens of postsynaptic densities (PSD) from rodent brains (Jordan et al, 2004; Yoshimura et al, 2004; Murata et al, 2005). PRR7 was found to interact with the membrane-associated guanylate kinase PSD95 via its C-terminal PDZ ligand and associate with the NMDAR complex (Murata et al, 2005). PRR7 structure resembles that of transmembrane adaptor proteins (TRAPs) (Hrdinka et al, 2011), which mediate T-cell receptor signaling by assembling a membrane proximal signalosome. In Jurkat T cells, the expression of PRR7 triggers apoptotic cell death that is preceded by c-Jun upregulation, suggesting that PRR7 might regulate T-cell survival in a manner that involves c-Jun (Hrdinka et al, 2011). Here, we show that NMDAR stimulation increases PRR7 levels in the nucleus and that PRR7 inhibits FBW7-mediated c-Jun ubiquitination. We propose that PRR7 is a neuronal FBW7 inhibitor and a novel nuclear signaling molecule linking NMDAR activity to c-Jun function. Moreover, we propose that PRR7 mediates NMDAR-mediated excitotoxicity in neuronal cultures in a c-Jun-dependent manner. Results PRR7 expression and subcellular distribution in brain Little is known about PRR7 expression in brain, so we first determined its tissue and developmental distribution. We found that PRR7 was highly expressed in rat forebrain (Appendix Fig S1A) and showed strong developmental regulation, with expression evident at postnatal day 7 and plateauing ~4 weeks after birth (Appendix Fig S1B). This was similar to the developmental profiles of the NMDAR subunit GluN1 and PSD95, which correlates with synaptogenesis in rodent brains. Immunocytochemistry of rat primary hippocampal cultures at DIV21 showed that endogenous PRR7 had a punctate distribution throughout synaptodendritic compartments and colocalized with synaptic markers (Fig 1A–C and Appendix Fig S1C). To determine the extent of synaptic colocalization, we performed triple colocalization analysis between PRR7, Bassoon, and PSD95 (Fig 1B) and found that 38.8 ± 3.6% (n = 9 neurons, P-value < 0.0001) of bona fide synapses (containing both PSD95 and Bassoon) contained PRR7. This suggests that PRR7 is abundant in some, but not all synaptic junctions. Exogenously expressed GFP-tagged PRR7 also colocalized with the synaptic marker Bassoon, corroborating imaging results of endogenous PRR7 distribution (Fig 1C). PRR7 was also present in the soma and showed variable expression in the nucleus (Fig 1A, lower panels). Quantitation of PRR7 abundance throughout the neuron showed enriched, but significantly variable expression in nuclear and synaptic regions compared to perinuclear and dendritic regions (Appendix Fig S1C). Western blot analysis of subcellular fractions isolated from rat brains showed that PRR7 was markedly enriched in the PSD fraction, but was also present in purified nuclear fractions (Fig 1D). Figure 1. Synaptic and nuclear localization of PRR7 Upper panels: Immunofluorescence imaging of PRR7 with Shank (synaptic) and MAP2 (dendritic) markers in DIV21 hippocampal neurons. Lower panels: single-plane Apotome images of nuclei (DAPI) show PRR7 can be present (arrow) or absent (arrowhead) from nuclei. Scale bar is 15 μm. Synaptic and dendritic distribution of PRR7 and pre (Bassoon)- and postsynaptic (PSD95) markers (arrowheads, triple colocalization). PRR7-GFP expressed in 17DIV hippocampal neurons is present in dendritic spines (arrows). Scale bar is 15 μm. PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (fibrillarin), SNAP25, and PSD95 were used as nuclear, presynaptic, and postsynaptic markers, respectively. Source data are available online for this figure. Source Data for Figure 1 [embj201593070-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint NMDAR activation elevates PRR7 expression in the nucleus PRR7 contains a putative lysine and arginine rich nuclear localization sequence (NLS) (KRR-RLR) near the N-terminus. Given immunocytochemical (Fig 1A and Appendix Fig S1C) and Western blot (Fig 1D) evidence of both synaptic and nuclear PRR7, we determined whether neuronal activity regulated the subcellular distribution of PRR7. Glutamate stimulation of rat DIV 21 primary hippocampal cultures increased PRR7 abundance in the nucleus (Fig 2A). This increase was also evident in the presence of the protein synthesis inhibitor cycloheximide, suggesting that it was not mediated by increased protein translation. We also noted that glutamate stimulation led to elevated levels of PRR7 in the soma (Fig 2A). However, the increase in nuclear PRR7 was larger than the increase in somatic PRR7 as measured by an elevated ratio of nuclear to cytoplasmic PRR7 following stimulation (Appendix Fig S2D). These results are consistent with an activity-dependent nuclear import. Glutamate stimulation also led to a concomitant reduction of PRR7 abundance at synapses that was not inhibited by the proteasome inhibitor epoxomicin (Fig 2B), suggesting that the decrease was not mediated by proteasomal degradation. To corroborate these results using another method, we treated high-density cortical cultures with glutamate and purified both synaptic and nuclear fractions from harvested cells (Appendix Fig S2A). As expected, glutamate stimulation led to an increase of PRR7 in the nucleus (Appendix Fig S2B) with a concomitant decrease at synapses (Appendix Fig S2C). To identify the specific ionotropic receptor activity required for this translocation, we treated neurons with glutamate in the presence of the NMDAR antagonist APV or the AMPAR antagonist CNQX. Only APV blocked the increase in nuclear PRR7 (Fig 2C). Neither CNQX nor the voltage-gated sodium channel blocker tetrodotoxin (TTX) blocked this process, suggesting that the accumulation of PRR7 in the nucleus did not require action potentials. Figure 2. NMDAR activation regulates PRR7 subcellular distribution Immunocytochemical analyses show that glutamate stimulation (Glut, 100 μM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 μM) added 30 min prior to glutamate stimulation did not affect this increase (Cont = 100 ± 12.5%, Glut = 202.1 ± 15.2%; +cycloheximide Cont = 104.9 ± 3.6, Glut = 231.4 ± 10.9) (n = ˜30–50 cells). Values are presented as mean ± s.e.m. Statistical analysis was performed using the Mann–Whitney test. ***P < 0.001. Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont = 100 ± 3.6%, Glut = 32.3 ± 1.3%; +epoxomicin Cont = 99.8 ± 4.6, Glut = 42.9 ± 1.1) (n = 14–17 dendrites, > 1,200 puncta for each group). Values are presented as mean ± s.e.m. Statistical analysis was performed using the Mann–Whitney test. ***P < 0.001. Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 μM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 μM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 μM, tetrodotoxin], NMDA [50 μM, N-methyl-D-aspartate]) as indicated. Only APV treatment blocked the glutamate-dependent increase in nuclear PRR7 (108.1 ± 19%). Values are presented as mean ± s.e.m. Statistical significance was determined using one-way ANOVA in conjugation with Sidak post hoc test. (n = ∼30–50 cells from 3 independent experiments) *P < 0.05, ***P < 0.001, ****P < 0.0001. PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30 × 30 μm2) from optical sections (300 nm step size) of PRR7-GFP-expressing neurons (DIV17) before and after bleaching. Prior to stimulation, there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 μM) and TTX (1 μM) (lower panel). NMDA (50 μM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0′ (blue arrow in E). Scale bar is 15 μm. Quantification of changes in nuclear GFP fluorescent intensities was normalized to time point t0′ and corrected for bleaching. No significant differences in PRR7-GFP FRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up table (LUT). Data are represented as mean ± s.e.m.; n = 9 cells (NMDA) and n = 5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post hoc correction. ***P < 0.001. PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from green (488 nm) to red (568 nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568 nm) in the nucleus following NMDA stimulation (50 μM, 30 min). Scale bar is 15 μm. Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment (Average increase; NMDA = 617.8 ± 47.7%, n = 11 cells, P < 0.0005; MK801 = 206.1 ± 13.6%, n = 5 cells, n.s.). Values are presented as mean ± s.e.m. Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons. ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint To assess the dynamics of PRR7 redistribution, we used fluorescence recovery after photobleaching (FRAP) to track the nuclear accumulation of PRR7-GFP following NMDAR stimulation. PRR7-GFP was already present in the nucleus of non-stimulated cells with basal synaptic activity but was also prominently localized in dendrites and the soma (Figs 1C and 2D). We observed a rapid recovery of GFP fluorescence in photobleached nuclei in response to bath application of NMDA, indicating an activity-dependent nuclear import of PRR7 (Fig 2D and E). This import was not visible when we applied the NMDAR antagonist MK801 overnight and during stimulation (Fig 2D and E). Treatment with MK801 significantly decreased basal PRR7-GFP fluorescence in the nucleus and soma (Fig 2D). To confirm that PRR7 was translocating from distal dendrites into the nucleus, we transfected neurons with a PRR7 construct fused to the photoconvertible fluorescent protein mEos3 (Zhang et al, 2012b). Following expression of the fusion protein, we photoconverted PRR7-mEos in distal dendritic regions (ROIs) from green to red fluorescence and monitored accumulation of red-PRR7-mEos in the nucleus 30 min after stimulation with 50 μM NMDA (Fig 2F and G). We found a robust increase in red-PRR7-mEos in the nucleus following treatment with NMDA, but not with MK801 (Fig 2F and G). These results, together with FRAP experiments and imaging and biochemical analyses of endogenous PRR7, strongly suggest that PRR7 translocates from synapses to nuclei following NMDAR activity. PRR7 interacts with NMDARs in rat brain Several activity-dependent synapse-to-nucleus messengers bind to NMDARs (Jordan & Kreutz, 2009). We confirmed that PRR7 interacts with PSD95 and NMDARs in adult mouse brains (Murata et al, 2005) using co-immunoprecipitation experiments (Fig 3A). As PRR7 and NMDAR bind to different PDZ domains of PSD95 (Niethammer et al, 1996; Murata et al, 2005), we hypothesized that they might associate indirectly via PSD95. To clarify this, we first co-expressed GluN1 and a PRR7 construct with a C-terminal myc-tag blocking the PDZ ligand in HEK293 cells, which lack PSD95. However, PRR7 bound to GluN1 despite the blocked PDZ ligand, suggesting that PSD95 was not required (Fig 3B). This interaction was specific as PRR7 did not bind to the AMPAR subunits GluA1 or GluA2 (Fig 3C) and did not require the GluN2A or GluN2B subunits of NMDARs (Fig 3B). Binding to NMDARs was preserved under stringent conditions (RIPA buffer) and required the transmembrane domain (TM), the short N-terminus, and 15 amino acids of the intracellular domain of PRR7 (Fig 3D). Figure 3. PRR7 binding to GluN1 is regulated by NMDAR activity Co-Immunoprecipitations (Co-IPs) of PRR7 with GluN1, PSD95 and GluN2B from rat brain lysates. FT = flowthrough, IgG = normal IgGs. Co-IPs from HEK293 cells of C-terminal myc-tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95. Co-IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 (myc-tagged GluN2B and GluA2 and flag-tagged PRR7). (Top) Schematics of the different PRR7 constructs used for co-IP (FL = full length). (Bottom) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co-IPs performed as indicated. Co-IPs of PRR7 with GluN1 from high-density primary neuronal cultures following treatment with NMDA (100 μM, 30 min, representative of n = 4). Source data are available online for this figure. Source Data for Figure 3 [embj201593070-sup-0005-SDataFig3.zip] Download figure Download PowerPoint We next asked whether PRR7 might have any effect on NMDA-mediated currents in HEK cells (Appendix Fig S3A and B) or in primary hippocampal neurons (Appendix Fig S3C). We found that PRR7 had no effect on NMDAR currents when co-expressed with NMDAR subunits in HEK cells (Appendix Fig S3A and B), irrespective of expressing GluN1 with GluN2A (Appendix Fig S3A) or GluN2B (Appendix Fig S3B). Moreover, shRNA-mediated knockdown of PRR7 had no effects on NMDA currents in primary hippocampal neurons (Appendix Fig S3C). We also assessed possible effects of PRR7 on miniature AMPAR-mediated currents. However, overexpression of PRR7 in cultured neurons modified neither amplitude nor frequency of AMPAR-mediated currents (Appendix Fig S3E and F). Collectively, these data do not support a role for PRR7 in regulating synaptic function. As NMDAR activation resulted in an accumulation of PRR7 in the nucleus (Fig 2), we hypothesized that the interaction of PRR7 with GluN1 was modulated by NMDAR activity. We treated hippocampal cultured neurons with NMDA and found that this reduced the extent of PRR7-GluN1 interactions (Fig 3E). Together, our results suggest that PRR7 associates with GluN1 independent of PSD95 and dissociates from NMDARs following neuronal activity to allow for nuclear import. PRR7 inhibits c-Jun ubiquitination As PRR7 was shown to regulate c-Jun expression and phosphorylation (Hrdinka et al, 2011), we hypothesized that PRR7 could link NMDAR activity to c-Jun function. Therefore, we generated lentiviruses to knockdown (shRNAs) or overexpress PRR7 in primary neuronal cultures and found that PRR7 and c-Jun abundance were directly correlated. PRR7 knockdown decreased c-Jun abundance, while PRR7 overexpression increased c-Jun abundance (Fig 4A). Neither overexpression nor knockdown of PRR7 altered c-Fos abundance (Fig 4A). In HEK293 cells treated with cycloheximide, we found that PRR7 expression significantly increased c-Jun half-life (Fig 4B), suggesting that PRR7 inhibits c-Jun degradation. PRR7 contains a putative phosphodegron motif (VTPFLS) at amino acid 175, suggesting it may interact with the E3 ubiquitin ligase FBW7, which has been shown to ubiquitinate c-Jun in neurons (Nateri et al, 2004). To determine whether PRR7 regulated c-Jun ubiquitination, we expressed FBW7 in HEK293 cells in the presence or absence of PRR7 and performed ubiquitination pulldown assays. We chose HEK293 cells because they do not express PRR7, have low amounts of FBW7, and express high amounts of c-Jun. We confirmed that FBW7 overexpression increased c-Jun ubiquitination (Fig 4C) as previously reported (Nateri et al, 2004). However, co-expression of FBW7 with PRR7 significantly blocked c-Jun ubiquitination (Fig 4C). To determine whether PRR7 regulated c-Jun ubiquitination in neurons, we knocked down PRR7 in hippocampal neuronal cultures using lentiviral shRNAs and found that this led to increased c-Jun ubiquitination (Fig 4D). These results suggest that PRR7 inhibits c-Jun ubiquitination in neurons. PRR7 knockdown or overexpression had no effect on other FBW7 targets mTOR and Myc (Davis et al, 2014) in neurons (Fig 4E), or in HEK293 cells (Appendix Fig S4). Taken together, these results suggest that PRR7 specifically inhibits c-Jun ubiquitination and is not a general inhibitor of FBW7. Figure 4. PRR7 inhibits c-Jun ubiquitination Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. Values are presented as mean ± s.e.m. **P < 0.01. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n = 16 gels for c-Jun and 4 gels for c-Fos). Control or PRR7-transfected HEK293 cells were treated with cycloheximide for the indicated time points, and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% versus control 68.7 ± 7.9%) and 240 min (83.6 ± 5.2% versus control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n = 4 gels for each group performed blind). Values are presented as mean ± s.e.m. t-tests between similar time points were used to analyze results. **P < 0.01. PRR7, FBW7, and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n = 5 gels for all groups). Values are presented as mean ± s.e.m. Significance was determined using two-way ANOVA with Dunnett's post hoc test. *P < 0.05. Western blots of c-Jun ubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149 ± 10.62%, n = 6 gels) c-Jun ubiquitination level in comparison to control neurons (NT). Values are presented as mean ± s.e.m. Statistical significance was determined by t-test. **P < 0.01. Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc. Significance was determined using two-way ANOVA with Dunnett's post hoc test. Data are presented as mean ± s.e.m. All % values represent comparisons to control. **P < 0.01, ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 4 [embj201593070-sup-0006-SDataFig4.zip] Download figure Download PowerPoint PRR7 forms a complex with c-Jun and FBW7 To determine why PRR7 specifically inhibited c-Jun ubiquitination, we tested whether these proteins interact. We found that PRR7 co-immunoprecipitated with c-Jun when overexpressed in HEK293 cells (Fig 5A). Pulldown experiments in vitro using purified proteins demonstrated that these proteins interact directly (Fig 5B). The phosphodegron motif in PRR7 also suggested a direct interaction with FBW7. Indeed, PRR7 bound to FBW7 when overexpressed in HEK293 cells (Fig 5C), in vitro using purified proteins (Fig 5D), and also in rat brain lysates (Fig 5E). To determine whether PRR7 blocked c-Jun ubiquitination by simple competition for FBW7 binding, we examined the interaction between PRR7, FBW7, and c-Jun in HEK293 cells. Surprisingly, PRR7 and FBW7 bound more strongly to c-Jun when both were cotransfected into heterologous cells (Fig 5F), suggesting that a c-Jun/FBW7/PRR7 complex is stabilized in the presence of all components. This points to a more complex mechanism of inhibition rather than simple substrate sequestration. Figure 5. PRR7 interacts with c-Jun and the E3 ubiquitin ligase FBW7 Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7. Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun. Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. Pulldown assays in vitro using purified PRR7 and FBW7. Co-IPs of endogenous PRR7 and FBW7 from lysates of rat brain. Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled do" @default.
• W2502332015 created "2016-08-23" @default.
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• W2502332015 title "Synaptonuclear messenger <scp>PRR</scp> 7 inhibits c‐Jun ubiquitination and regulates <scp>NMDA</scp> ‐mediated excitotoxicity" @default.
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