KLHL21 Negatively Regulates The NF-?B Signaling Through Suppressing IKK? Kinase

The transcription factor NF-?B(nuclear factor of kappa B)plays a pivotal role in innate immune responses,cell survival and many other biological processes.In the resting cells,NF-?B is sequestered in the cytoplasm by binding to members of the I?B(inhibitor of kappa B)proteins,which mask its nuclear localization signal(NLS).When cells were exposed to extracelluar stimuli such as TNFa(tumor necrosis fector),TRAF2(TNF receptor-associated fector 2)rapidly recruited TAKI(TGF? activated kinase 1)to TNFRl(tumor necrosis fector receptor 1).Meanwhile,the IKK(I?B kinase)complex was also directly recruited to TNFR1 signaling complex by TRAF2.After the assembly of all of these signaling proteins,the TAK1 catalyzed the phosphorylation and activation of IKK complex.The I?B? was then phosphorylated by the activated I?B kinase,polyubquitinated by a ubiquitin ligase complex,and degraded by the 26S proteasome.Then NF-?B was released from I?B?,and translocated from the cytoplasm into the nucleus and initiated the transcription of its target genes.The I?B kinase(IKK)complex,consists of the protein kinases IKKa and IKK?and a regulatory component called IKKy(NEMO).Phosphorylation of IKKa/IKK?is essential for the activation of IKK kinase complex,and subsequent phosphorylation and ubquitatination of I?B?,and the activation of the canonical NF-?B signal pathway.IKK kinase has also been reported to play important roles in the occurrence and development of tumors,cell proliferation and apotosis.Deciphering its mechanism in cells may also provide potential therapeutic targets and novel strategies for treating inflammation-related diseases in chnic.There are 42 members of KLHL(Ketch-like)protein family encoded in the human genome,which consist of a BTB domain,a BACK domain,and five to six Kelch motifs.They are highly conserved in evolution.So far,the specific roles for most of the family members have not yet been elucidated.But some of the KLHL family proteins have been reported to interact with cullin3 protein to form functional E3 ubquitin ligase complex,and catalyze the ubiquitin modification of their subrate proteins.KLHL21 is a member of KLHL family,which can also interact with cullin3 to form E3 ubiquitin ligase complex and mediated the ubiquitination of Aurora B in vitro.It is essential for cytokinesis and regulates the translocation of the chromosomal passenger complex from chromosomes to the spindle midzone in anaphase.Our preliminary work identified that KLHL21 could interact with IKK? kinase.It inhibited the phosphorylation and degradation of I?B? induced by TNF? and repressed the translocation to nuleus of p65(Red A).But the mechanism of its interaction with IKK? protein and its functional implication in NF-?B signal pathway are still unclear.Thus,the aim of this work is to analyze the function and regulatory mechanism of KLHL21 in the NF-?B signaling pathway.Firstly,further confirmed the interaction between KLHL21 and IKK? protein.First,the endogenous interaction between IKK? and KLHL21 in HEK293T and RAW264.7 cells was confirmed by co-immunoprecipitation Second,GST-KLHL21 fusion protein was prokaryotically expressed and purified,GST pull-down assay was applied to analyze the interaction between GST-KLHL21 and endogenously expressed IKK? in HEK293T and RAW264.7 cells.Last,immunofluorescence co-location assay also showed the colocalization of endogenous IKK? and mCherry tagged KLHL21 in HeLa cells.These results clearly confirmed that KLHL21 could interact with IKK(3 both in vivo and in vitro.To farther analyze whether KLHL21-IKK? interaction was influenced by external stimuli such as TNFa,HEK293T cells were transiently co-transfected with vectors encoding C-terminal EE-tagged IKK?(IKK?-EE)and C-terminal 3xFLAG-tagged KLHL21(KLHL21-3F).24 h post-transfection,the cells were treated with 20ng/ml TNFa for 0 min,10 min and 30 min respectively.The immunoprecipitation results revealed that the interaction between KLHL21 and IKK? was gradually attenuated in response to TNFa treatment.Secondly,identification of the interaction domains in IKK? and KLHL21.IKK? protein possesses a KD(kinase domain)domain,a ULD(ubiquitin like domain)domain,a SDD(scaffold/dimerization domain)domain and a NBD(NEMO-binding domain)domain.To identify which domain of IKK? mediated its interaction with KLHL21,five deletion mutants of IKK? have been constructed.HEK293T cells were cotransfected with full-length IKK?,IKK? deletion mutants and wild-type KLHL21 respectively,Co-immunoprecipitation analysis results revealed that IKK? interacted with KLHL21 through its KD domain We have also constructed four deletion mutants of KLHL21 and one point mutant KLHL21M(D114A/L115A/Q117A,the mutation sites located in the BTB domain,which could not bind to cullin3).Then HEK293T cells were co-transfected with WT KLHL21,KLHL21 deletion mutants and WT IKK(3 respectively.The Kelch domains of KLHL21 were found to be required for its interaction with IKK? by co-immunoprecipitation assay.KEAP1,another member of KLHL protein family,has been reported to bind to the E(T/S)GE motif in the KD domain of IKK? through its Kelch domain.To ananlyze whether KLHL21 could also bind to this specific motif by its Kelch domain,we mutated the E39 of the motif into alanine.Co-immunoprecipitation assay results revealed E39A mutation of IKK? almost completely aborted its interaction with the KEAP1,which did not affect its interaction with KLHL21.This result suggested that KLHL21 may bind to other motifs in the KD domain of IKK?,and further revealed the interation between them was specific.Thirdly,KLHL21 suppressed the activation of IKK?.To analyze whether KLHL21 could suppress the activation of IKK? in vivo,HEK293T cells were transiently co-transfected with HA-ubiquitin,IKK?-FLAG,KLHL21-EE and KLHL21M-EE respectively.The cells were treated with proteasome inhibitor MG132(5 ?mol/L)for 16 h.Western blotting results revealed that both KLHL21 and KLHL21M could not promote the ubiquitination of IKK?.This suggested that KLHL21 was not the specific E3 ubquitin ligase of IKK? in cells.Instead,coexpression of KLHL21 and KLHL21M suppressed the autophosphorylation of IKK? induced by overexpression.And overexpression of KLHL21 also significantly suppressed the phosphorylation of endogenous IKK?/? in HEK293T cells upon 20 ng/ml TNF? treatment.Consistent with this,knocking-down KLHL21 with specific siRNAs in HEK293T cells enhanced the phosphorylation of IKK?/? in response to TNFa treatment.These results clearly revealed KLHL21 could suppress the activation of IKK? independent of its E3 ubiquitin ligase activity.To analyze whether KLHL21 binding to IKK? interfered its interaction with TRAF2,TAK1 or I?B?,KLHL21 and IKK? were transiently co-expressed in HEK293T cells.Co-immunoprecipitation assay results revealed that overexpressing KLHL21 didn't interfere the interaction between IKK? and TRAF2,TAK1 or I?B?.Lastly,KLHL21 differentialy regulated the expression of NF-?B target genes.To analyze the effects of overexpressing KLHL21 on NF-?B signal pathway,HEK293T cells transiently transfected with KLHL21 were stimulated with 20ng/ml TNF? and harvested at different time points(0 h,0.5 h,2 h,4 h).The expression of NF-?B target genes were analyzed by real-time PCR.Overexpressing KLHL21 significantly suppressed TNF? induced NFKBIA(encoding I?B?)transcription Unexpectedly,it further upregulated the induced expression of IL-8(2 h and 4 h)and TNFAIP3(4 h).Knocking-down KLHL21 with specific siRNA significantly upregulated the expression of NFKBIA while it downregulated the expression of IL-8(2 h and 4 h).However,it had no obvious effects on the induction of TNFAIP3.Moreover,knockdown of KLHL21 with siRNA also upregulated the expression of NFKBIA,while it downregulated the expression of TNFAIP3 and IL-1B in RAW264.7 cells uponLPS treatment.These results revealed KLHL21 differentially modulates the expression of NF-?B target genes.To analyze the reason of this phenomenon,HEK293T cells were transiently transfected with KLHL21-3F.24h post-transfection,the cells were treated with 20 ng/ml TNF? and harvested at different time points(0 h,2 h,4 h).The protein level of p65 in nucleus was analyzed by western blotting.The result revealed overexpressing KLHL21 extended duration of p65 in nucleus(4 h),which may account for the upregulated the expression of relative late genes such as IL-8 and TNFAIP3 in HEK293T cells upon TNF?treatment.In conclusion,KLHL21 can specifically bind to the kinase domain of IKK?through its Kelch domain.It inhibited the activation of IKK? and differently regulated the NF-?B target genes in response to TNF? treatment.In this work,we have achieved following results:1.Endogenously expressed IKK? interacted with KLHL21 protein in both HEK293T and RAW264.7 cells.GST-pull down assay futher confirmed the interaction between KLHL21 and IKK? protein which suggested they may interact directly in vivo.Immunofluorescence staining also showed the colocalization of endogenous IKK? and KLHL21 in HeLa cells.And the interaction between KLHL21 and IKK? was gradually attenuated in response to TNFa treatment.2.The IKK? deletion mutants and KLHL21 deletion mutants have been successfully constructed.Co-immunoprecipitation assay revealed that IKK?bound to the Kelch domain of KLHL21 by its KD domain.IKK? E39A mutant was successfully constructed.Co-immunoprecipitation assay revealed that KLHL21 could not bind to E(T/S)GE motif in the KD domain of IKK? in cells like KEAP1.It might bind to other sites in the KD domain.These results further demonstrated the interaction between them was specific.3.Co-overexpression of KLHL21 and KLHL21M foiled to enhance the ubiquitination of IKK?,which demonstrated KLHL21 was not a specific E3 ubiquitin ligase of IKK? in cells.Instead,overexpressing KLHL21 and KLHL21M dramatically inhibited the auto-phosphorylation IKK? induced by overexpression And KLHL21 also inhibited the phosphorylation of IKK?/? in HEK293T cells upon TNFa treatment.While knocking-down KLHL21 with specific siRNAs enhanced the phosphorylation of IKK?/? in response to TNFa treatment,it did not affect protein level of endogenous IKK?.4.Overexpressing KLHL21 did not interfere with the interaction between IKK?and TRAF2,TAK1 or IKB? respectively.5.Overexpressing KLHL21 significantly suppressed TNFa induced NFKBLA transcription,while it upregulated the induced expression of IL-8(2 h and 4 h)and TNFAIP3(4 h)in HEK293T cells.Knocking-down KLHL21 with siRNA in HEK293T cells significantly upregulated the expression of NFKBIA,while it downregulated the expression of IL-8(2h and 4h).But it had no obvious effects on the induction of TNFAIP3 And knocking-down KLHL21 also upregulated the expression of NFKBLA,and downregulated the expression of TNFAIP3(4 h)and IL-1B(0.5 h and 4 h)in RAW264.7 cells upon LPS treatment.Further analysis revealed KLHL21 differentialy regulated NF-?B target genes may be due to the fact that overexpressing KLHL21 extended the duration of p65 in nucleus(4 h)in HEK293T cells upon TNFa treatment.