The Deubiquitinating Enzyme USP48 Regulates TNF? Signaling By Stabilizing TRAF2

Background In acute infectious diseases or a systemic inflammatory response,tumor necrosis factor ?(TNF?)is highly upregulated and epithelial layers of organ tissues lose barrier integrity,causing fluid handling at the tissue level and impairing organ function.At the cellular level,TNF? signaling results in loss of E-cadherin.Loss of E-cadherin disrupts establishment of cell-cell junction and results in increases in epithelial permeability and motility.Mechanistic insights into E-cadherin regulation could greatly enhance our understanding of the pathogenesis and approach to treatment human disorders caused by epithelial barrier dysfunction.The tumor necrosis factor receptor-associated factor 2(TRAF2)is a second messenger adaptor protein that plays an essential role in propagating TNF?-mediated signaling pathways.Upon TNF? stimulation,TRAF2 is recruited to TNFR1 or TNFR2,leading to the activation of NF-?B and c-Jun N terminal kinase(JNK)signaling pathways.While JNK has been shown to play important role in regulating E-cadherin junctions,the effect of TRAF2 on E-cadherin expression has not been studied.Modulation of TRAF2 activity by ubiquitination is well studied;however,the deubiquitinating enzyme(DUB),which regulates TRAF2 stability,has not been identified.Recently,the USP48 DUB,also known as USP31,has been shown to associate with TRAF2 without any description of USP48 action on TRAF2 activity or stability.USP48 is widely expressed in all normal tissues,suggesting that it participates in multiple essential deubiquitinating reactions.However,the physiologic function of USP48 remains largely unknown,we want to test the functional significance of this interaction.Methods 1.USP48 affects TRAF2 protein levels were analyzed by western blotting;USP48 affects TRAF2 m RNA levels were analyzed by q RT-PCR.2.USP48 affects ubiquitinated TRAF2(Ub-TRAF2)levels were performed by cellular ubiquitination assays;USP48 is associated with TRAF2 were analyzed by Co-immunoprecipitation;The cellular location of TRAF2 and USP48 were detected by immunostaining. 3.TNF? and GSK3? phosphorylates USP48 were analyzed by immunoprecipitation;USP48 is associated with GSK3?were analyzed by Co-immunoprecipitation;The cellular location of GSK3? and USP48 were detected by immunostaining.4.GSK3? affects TRAF2 protein levels were analyzed by western blotting;GSK3? affects ubiquitinated TRAF2(Ub-TRAF2)levels were performed by cellular ubiquitination assays.5.The impact of USP48 phosphorylation on hydrolysis of K-48-diubiquitin were performed by an in vitro DUB assay;USP48 wild type protein,USP48C98 S mutant protein,and USP48?886-890 mutant protein were synthesized in a reticulocyte lysate transcription and translation(Tn T)system.6.USP48,JNK,TNIK affects E-cadherin expression were analyzed by western blotting;USP48,JNK,TNIK affects E-cadherin m RNA levels were analyzed by q RT-PCR.7.USP48,JNK,TNIK affects TEER were used an electric cell-substrate impedance sensing(ECIS)system.Results 1.USP48 stabilizes TRAF2.1)Knockdown or Inhibition of USP48 decreases TRAF2 levels.2)Knockdown of USP48 has no effect on TRAF2 m RNA levels.3)USP48 overexpression increases TRAF2 half-life.2.USP48 deubiquitinates TRAF2 in the cytoplasm.1)Overexpression of USP48 reduces TRAF2 ubiquitination.2)Inhibition of USP48 activity elevates TRAF2 ubiquitination.3)Knockdown of USP48 increases K-48-linked polyubiquitination of TRAF2.4)USP48 is associated and co-localized with TRAF2.3.TNF? induces USP48 site specific phosphorylation,which is mediated by GSK3?.1)TNF? and GSK3? phosphorylates USP48.2)USP48 has a GSK3? consensus phosphorylation motif(amino acid 886-890)3)Inhibition of GSK3? attenuates TNF?-induced phosphorylation of USP48.4)USP48 is associated and co-localized with GSK3?.4.USP48 phosphorylation is required for TRAF2 ubiquitination and stability.1)Inhibition of GSK3? promotes TRAF2 degradation.2)Inhibition of GSK3? promotes TRAF2 ubiquitination. 3)Overexpression of GSK3?S9A reduces TRAF2 ubiquitination.4)Phosphorylation motif mutant of USP48 loses effect of USP48 on stabilization of TRAF2.5)Overexpression of GSK3? phosphorylation motif mutant of USP48 increases TRAF2 ubiquitination.5.Phosphorylation of USP48 by GSK3? increases USP48 activity.1)Inhibition of GSK3? or deletion of phosphorylation motif of USP48 has no effect on association between USP48 and TRAF2.2)Inhibition of GSK3? attenuates USP48 effect on deubiquitination of TRAF2.3)Phosphorylation motif deletion mutant of USP48 loses DUB activity.4)GSK3?S9A increases USP48 DUB activity.6.Knockdown of USP48 attenuates TRAF2-dependent activation of JNK1,therefore increasing E-cadherin expression.1)Knockdown of USP48 attenuates TNF?-induced phosphorylation of JNK1 and c-Jun.2)Knockdown of USP48 increases E-cadherin expression.3)Inhibition of JNK attenuates TNF?-induced reduction of E-cadherin.4)Inhibition of TNIK attenuates TNF?-induced reduction of E-cadherin.5)Knockdown of USP48 attenuates TNF?-induced reduction of E-cadherin.6)Knockdown of USP48 increases E-cadherin m RNA levels.7)Inhibition of JNK or TNIK increases E-cadherin m RNA levels.7.Knockdown of USP48 or inhibition of TNIK and JNK promotes epithelial barrier integrity.1)Knockdown of USP48 increases TEER.2)Inhibition of TNIK or JNK increases TEER.3)Knockdown of USP48 promotes LPA-increased TEER.Conclusion In conclusion,we provide a new framework for studying TRAF2 protein stabilization.In our cellular model system,GSK3? phosphorylates USP48 resulting in increased activation of USP48 which deubiquitinates TRAF2 to enhance TRAF2 stability and TNF?-mediated JNK signaling.USP48 deficiency attenuates TNF?-induced JNK activation,thereby increasing E-cadherin expression and epithelial barrier integrity.This GSK3?–USP48?TRAF2?TNIK?JNK?E-cadherin cascade may play an important role in cellular responses and could represent a new pharmacologic target that partially suppresses TNF? signaling through TRAF2/JNK pathway and enhances epithelial barrier integrity in the setting of acute illness.