title: Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction.
abstract: BACKGROUND
  Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14's catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14's catalytic activity is required for neuromuscular junction (NMJ) structure and function.
  
  
  RESULTS
  Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice.
  
  
  CONCLUSIONS
  USP14's catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center, Evelyn F. McKnight Brain Institute, 1825 University Blvd, Birmingham, AL, 35294, USA. jjh85@uab.edu.
authors.forename: Jada H
authors.initials: JH
authors.lastname: Vaden
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, 303 East Chicago Ave, Chicago, IL, 60611, USA. bula@northwestern.edu.
authors.forename: Bula J
authors.initials: BJ
authors.lastname: Bhattacharyya
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Danny Thomas Place, Memphis, TN, 38105-3678, USA. Ping-Chung.Chen@stjude.org.
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Chen
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center, Evelyn F. McKnight Brain Institute, 1825 University Blvd, Birmingham, AL, 35294, USA. jawatson@uab.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Watson
authors.affiliation: Department of Physiology, Neurobiology and Behavior, University of California, Davis, California, CA, USA. agail22@uab.edu.
authors.forename: Andrea G
authors.initials: AG
authors.lastname: Marshall
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center, Evelyn F. McKnight Brain Institute, 1825 University Blvd, Birmingham, AL, 35294, USA. scottep@uab.edu.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Phillips
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center, Evelyn F. McKnight Brain Institute, 1825 University Blvd, Birmingham, AL, 35294, USA. wilsonja@uab.edu.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Wilson
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center, Evelyn F. McKnight Brain Institute, 1825 University Blvd, Birmingham, AL, 35294, USA. gdking@uab.edu.
authors.forename: Gwendalyn D
authors.initials: GD
authors.lastname: King
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, 303 East Chicago Ave, Chicago, IL, 60611, USA. r-miller10@northwestern.edu.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Miller
authors.affiliation: Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center, Evelyn F. McKnight Brain Institute, 1825 University Blvd, Birmingham, AL, 35294, USA. wilson@nrc.uab.edu.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Wilson
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D001259:Ataxia / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D002384:Catalysis; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D005106:Exploratory Behavior; D005260:Female; D018737:Hand Strength; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008818:Mice, Neurologic Mutants; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D009469:Neuromuscular Junction / Q000503:physiopathology* / Q000648:ultrastructure; D009474:Neurons / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011758:Pyrroles / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D045442:Rotarod Performance Test; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D019076:Transgenes; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D054875:Ubiquitination
chemical_list: C000594744:1-(1-(4-fluorophenyl)-2,5-dimethylpyrrol-3-yl)-2-pyrrolidin-1-ylethanone; D000873:Anthracenes; D009419:Nerve Tissue Proteins; D011758:Pyrroles; D011759:Pyrrolidines; C469268:Usp14 protein, mouse; C432165:pyrazolanthrone; D048031:JNK Mitogen-Activated Protein Kinases; D043222:Ubiquitin Thiolesterase; D046988:Proteasome Endopeptidase Complex