HuangandHungJournalofBiomedicalScience2013,20:3 http://www.jbiomedsci.com/content/20/1/3 REVIEW Open Access κ κ Beyond NF- B activation: nuclear functions of I B α kinase Wei-Chien Huang1,2,3,4* and Mien-Chie Hung1,2,3,4,5* Abstract IκB kinase (IKK) complex, themaster kinasefor NF-κBactivation, contains two kinase subunits, IKKα and IKKβ.In addition to mediating NF-κBsignaling by phosphorylating IκBproteins during inflammatoryand immune responses, the activation of theIKK complex also responds to various stimuli to regulate diverse functions independently ofNF-κB. Although thesetwo kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβaccount for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic,IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles ofIKKα have broughtincreasing complexity to itsbiological function. Thisreview highlights majoradvances inthe studies of thenuclear functions of IKKα and themechanisms ofIKKα nuclear translocation. Understanding thenuclear activityis essential for targeting IKKα for therapeutics. Keywords: Nuclear IKKα, NF-κB, Gene transcription, Tumor progression Introduction Although IKKα and IKKβ share structural and bio- IκB kinase (IKK)/Nuclear factor kappa B (NF-κB) family chemicalsimilarities,differentphenotypesbetweenIKKα signaling mediates the expression of hundreds of genes and IKKβ knockout mice imply distinct physiological involved in inflammation, immune response, cell survival, roles of the IKK isoforms [6]. IKKβ deficiency results in and cancer [1-4]. NF-κB proteins are part of a molecular embryonal death and shows the defective response to cascade that begins with signals outside the cell and cul- inflammatory cytokines andlivercellapoptosis[7]. IKKα minates in the nucleus by binding to DNA and activating knockout mice display the defective proliferation and gene expression. The best-known form of NF-κB consists differentiation of kerationocyte and the abnormalities of ofthe DNA-bindingsubunit p50 and a transcription acti- limb and skeleton, suggesting the requirement of the vator,p65(alsoknownasRelA).Intheabsenceofspecific IKKα subunit in morphogenesis [8]. Importantly, these extracellular signals, NF-κB inhibitors, such as IκB, p105, studies of gene knockout have shown that IKKα is dis- and p100 proteins, tether to NF-κB in the cytoplasm to pensable for IκB degradation although both IKKα and prevent NF-κB-mediated gene transcription [1]. When IKKβ are critical for NF-κB-mediated gene expressions. cells receive appropriate stimuli, such as tumor-necrosis Instead of its role in phosphorylating IκBα in classic NF- factor-α (TNF-α), a ternary IKK complex consisting of κB activation, IKKα homodimer has been shown to IKKα, IKKβ and NEMO (IKKγ) induces IκB phosphoryl- mediate the processing of p100 precursor to p52 by the ation, leading to IκB ubiquitination and proteasomal noncanonical NF-κB pathway [5]. IKKα- and IKKβ- degradation that are required for liberation of NF-κB in deficient mouse embryo fibroblasts exhibit different the nucleus where it binds to specific promoter elements patterns of β-catenin cellular localization in which IKKβ toactivategeneexpression[1,5]. decreases β-catenin-dependent transcriptional activation while IKKα increases β-catenin-dependent transcriptional activity [9]. Differential requirements for IKKα and IKKβ *Correspondence:[email protected];[email protected] werealsofoundinprimaryhumanosteoarthriticchondro- 1CenterforMolecularMedicine,ChinaMedicalUniversityHospital,Taichung cytedifferentiation[10]. 40447,Taiwan 2GraduateInstituteofCancerBiology,ChinaMedicalUniversity,No.6Hsueh- In addition to phosphorylating distinct substrates in HsihRoad,Taichung404,Taiwan the cytoplasm [6,11], sub-cellular distribution of IKKα is Fulllistofauthorinformationisavailableattheendofthearticle ©2013HuangandHung;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. HuangandHungJournalofBiomedicalScience2013,20:3 Page2of13 http://www.jbiomedsci.com/content/20/1/3 also different from that of IKKβ, further indicating that predominantlycytoplasmicIKKβ,IKKαhasbeendetected these two related signaling kinases are functionally dif- in both the nucleus and the cytoplasm of MEF cells at ferent. Many studies have indicated that IKKα can be resting state [9,25]. The constitutive shuttling of IKKα detected in both the cytoplasm and the nucleus whereas between cytoplasm and nucleus was furtherconfirmed by IKKβ is detected predominantly in the cytoplasm [12-16]. accumulation of IKKα in the nucleus of Hela cells in the The observation of nuclear/cytoplasm shuttling of IKKα presenceofanuclearexportblockingagent,leptomycinB led to the discovery of the first nuclear role of IKKα in (LMB)[13].Therefore,theproteinbindingandphosphor- phosphorylating histone H3, which results in NF-κB- ylation of specific pools of β-catenin by IKKα in the mediated gene expression [12,16]. These studies provided nucleus have been proposed to explain the contradictory an explanation of why IKKα is dispensable for IκBα effect of the IKK isoforms on β-catenin-dependent tran- degradation but remains essential for NF-κB-dependent scription [9]. However, the phosphorylation sites and transcription. Aside from nuclear regulation of NF-κB- detailedmechanismsthataccountforthedifferentialregu- dependent gene transcription through chromatin modifi- lationofβ-cateninbyIKKisoformsremaintobeexplored. cation in response to pro-inflammatory stimuli, nuclear IKKcomplexisactivatedinresponsetovariousstimuli IKKα also functions in apoptosis, cell cycle, and tumor involving inflammation, apoptosis, immune response, progressionincolorectal[17,18],breast[19,20],pancreatic and cancer. These physiological and pathological stimuli [21], gastric [22], osteosarcoma [23], and prostate [24] have been reported to enhance the nuclear levels of cancers.The currentunderstandingsofnucleartransloca- IKKα (Figure 1). TNF-α, a critical pro-inflammatory tionandfunctionsofIKKαarediscussedbelow. cytokine, was the first stimulant for IKKα nuclear trans- location identified [12,16]. Other inducers of alterative NuclearIKKαregulatesNF-κB-dependentgene NF-κB pathway, including lymphotoxin β and CD40, transcriptionandinflammation also elicit nuclear IKKα signaling [15]. In addition to Nuclear expression and functions of IKKα were first dis- TNF-α, Helicobacter pylori (HP) can also trigger nuclear covered based on the observation of different patterns of translocation of IKKα via its virulence factor CagA protein β-catenin activation in IKKα- and IKKβ-deficient mouse to induce cytokine production for appropriate inflamma- embryonic fibroblast (MEF) cells by Lamberti et al. in tory responses [22]. Similarly, overexpression of Kaposi’s 2001 [9]. Their study showed β-catenin-dependent tran- sarcoma-associated herpesvirus (KSHV)-encoded viral scription was decreased by IKKβ but increased by IKKα FLICE inhibitory protein K13 [26] and hepatitis B virus- that is likely due to the differential abilities of IKKα and encoded X (HBx) protein [14] also induce IKKα nuclear IKKβ to bind to and phosphorylate β-catenin. Unlike the translocation to regulate NF-κB activity. The requirement Figure1NuclearIKKα-dependentmolecularregulationsofNF-κB-mediatedgenetranscription.Inresponsetoavarietyofstimuli, includingproinflammatorycytokines,pathogens,andgrowthfactors,IKKαtranslocatesintothenucleusandbindtoDNAinassociationwithCBP tophosphorylatehistoneH3atSer10,CBPatSer1382/1386,andp65atS536.NuclearIKKαalsoremovesrepressiveHDAC3/SMRTcomplexfrom NF-κB-dependentgeneexpressionthroughphosphorylatingSMRTatSer2410.Theseeventsfacilitatetheformationoftranscriptional enhanceosometoincreaseNF-κB-dependentgeneexpression.Ontheotherhand,nuclearIKKαalsocontributestotheterminationofNF-κB- mediatedgenetranscriptionsbyphosphorylatingp65atSer536andPIASatSer90tofacilitatetheturnoverofp65inresponsetoTNF-αorLPS stimulation. HuangandHungJournalofBiomedicalScience2013,20:3 Page3of13 http://www.jbiomedsci.com/content/20/1/3 ofcytoplasmic/nuclearshuttlingandchromatinassociation and phosphorylates its HAT domain at Ser1382 and of IKKα for NF-κB-dependent gene regulation in aTAK1- Ser1386 to enhance the enzymatic activity of CBP on dependent manner in TLR4-activated antigen-presenting histone acetylation [15](Figure 1).CBPanditshomolog, cells[27]andactivatedneutrophils[28,29]furthersupport p300, are transcriptional coactivators that function in that nuclear IKKα functions as a common and important the communication between transcription factors and regulator for NF-κB activity in response to various im- the transcriptional machinery [33]. Since the availability muneandinflammatorystimuli(Figure1). of these coactivators is limited due to their essentiality As illustrated in Figure1,TNF-α-inducednuclear IKKα for large number of transcription factors, competition mediates NF-κB-dependent gene transcription, regardless between different transcription factors for CBP or p300 of IκBα degradation, by enhancing transactivation [25] has been proposed to play a role in the coordination of andDNAbinding[30]ofp65aswellaschromatinregula- gene expression and the appropriate execution of many tion through its interaction CREB-binding protein (CBP), biological processes [34]. Our study further demon- a histone acetyltransferase [15,16]. Phosphorylation of strated that the IKKα-dependent CBP phosphorylation serine residues within the transactivation domains (TA1 enhances NF-κB-mediated gene expression and sup- and TA2) of p65 is responsible for transcriptional activa- presses p53-mediated gene expression by switching the tion of the NF-κB target genes [31]. By employing the binding preference of CBP from p53 to NF-κB, thereby Gal4 DNA binding domain fused to the p65 transactiva- promoting cell growth [15]. Similar to IKKα, IKKγ/ tion portion in a heterologous luciferase reporter assay, NEMO has also been shown to shuttle between the nuclear IKKα was shown to transduce NF-κB-inducing cytoplasm and the nucleus and to compete with p65 and kinase (NIK)-dependent p65 TA1 transcriptional activity, IKKα for binding to the N-terminus of CBP. Even suggesting that IKKα phosphorylates the TA1 domain of though IKKγ/NEMO is essential for the kinase activity p65 in the nucleus [25]. In addition, using the IKKα of IKK complex in the cytoplasm, it seems to act as a mutant lacking an intact nuclear localization sequence negative regulator of nuclear IKKα and inhibit CBP- (NLS),p65chromatinimmunoprecipitationassaysfurther dependenttranscriptionalactivationinthenucleus [35]. revealed thatnuclear IKKα plays a role inbindingactivity CBP and p300 mediate acetylation of histones as well ofNF-κB/p65tosomebutnotallNF-κB-targetpromoters as many transcription factors, including p65, for their by removing histone deacetylase 3 (HDAC3), a negative transcriptional potential [33]. Acetylation of p65 is crit- regulator of NF-κB-dependent transcription, from these ical for its DNA binding and transactivation activity promoters [30]. Even though several earlier studies have [36,37]. In contrast, deacetylation of p65 by HDACs, in- implicated the function of IKKα in chromatin, the role of cluding HDAC1, HDAC2, HDAC3, and SIRT1, has been nuclear IKKα as a chromatin modifier was not reported reported to repress its transcriptional activity [38,39]. until later by Baldwin’s and Gaynor’s group [12,16]. They The enzymatic activities of HDACs are regulated by their showed that IKKα functions as a chromatin kinase in the ability to associate with co-repressor proteins, such as nucleus and targets histone H3 at Ser10 for activation of SMRT and NCoR [40]. Chromatin-associated HDAC3/ NF-κB-directed gene expression (Figure 1). The nuclear SMRTcomplex tethered by p50 homodimers on NF-κB- import,chromatinassociation,histonephosphorylationof target cIAP and IL-8 promoters of unstimulated cells is IKKα relies on its kinase activation by the upstream NIK responsible for the basal suppression of NF-κB-regulated kinaseinresponsetobothTNF-αandendotoxinlipopoly- gene transcriptions [18]. In addition to enhancing histone sarccharide (LPS) [32]. However, the correlation between acetylation by targeting CBP, IKKα was also found to re- chromatin-bound IKKα and histone H3 phosphorylation lieve the suppression of NF-κB-mediated transcription by on NF-κB-target genes was not found in human prostate removingtheHDAC3/SMRTrepressorcomplexfromtar- carcinomaDU145 cells[18],indicatingthatcelltype- and getpromoters[18,30,41].Chromatinimmunoprecipitation target gene-specificity exists in IKKα-dependent histone analysis demonstrated that upon attachment to laminin, phosphorylation. the induction of chromatin-associated IKKα protein and Gaynor’s group further showed that nuclear IKKα acetylated histone is accompanied by a decrease in interacts with the transactivation domain of CBP. The chromatin-bound HDAC3/SMRT complex. Direct phos- IKKα/CBP complex in conjunction with p65 is recruited phorylation of SMRT at Ser2410 by IKKα on chromatin to the NF-κB responsive promoters to mediate cytokine- also stimulates nuclear export and proteasomal degrad- induced phosphorylation and subsequent acetylation of ation of the HDAC3/SMRTcomplex by recruiting TBL1/ specific residues in histone H3 [16]. These findings TBLR1,Ubc5,and14-13-3εproteins[18].Theremovalof suggested that IKKα not only targets on NF-κB but also HDAC3/SMRT by IKKα allows the active p50-RelA/p65 functions as a key epigenetic regulator to initiate se- heterodimerofNF-κBtobindandpotentiatetranscription quential chromatin modifications. Our studies also (Figure1).AlthoughtheSMRTcorepressorreturnstothe demonstrated that nuclear IKKα binds directly to CBP chromatin-bound NF-κB complex almost immediately HuangandHungJournalofBiomedicalScience2013,20:3 Page4of13 http://www.jbiomedsci.com/content/20/1/3 after the active p50-RelA/p65 heterodimer binds to the p65 Ser536 phosphorylation and other regulatory factors promoter, IKKα remains associated with the chromatin arerequired.TheworkbyLiuet al.furtherdemonstrated and phosphorylates both p65 at Ser536 and SMRT at thatproteininhibitorofactivatedSTAT1(PIAS1),agene- Ser2410topreventtherecruitmentandchromatinassoci- specific transcriptional repressor with SUMO E3 ligase ation of HDAC3 at the NF-κB-regulated promoter. activity,isinvolvedintheIKKα-mediatednegativeregula- Displacement of HDAC3 from active NF-κB allows p300 tion of NF-κB and inflammation restriction [44]. In toloadandsubsequentlyacetylatep65atLys310,whichis response to various inflammatorystimuli, includingTNF- required for full NF-κB transcription [41]. Thus, IKKα- α and LPS, PIAS is rapidly phosphorylated at Ser90, and mediated derepression of SMRT is an initial step critical this phosphorylation is mediated by IKKα and required for NF-κB transcription and survival in response to for its association with chromatin and enzymatic activity laminin attachment. Interestingly, cigarette smoke extract to repress promoter-binding and transcriptional activities (CSE)wasrecentlyreportedtoinducethetranslocationof of NF-κB [44]. It would be interesting to further address IKKαfromcytoplasmtonucleusinmouselungtissueina the relationship between p65 Ser536 phosphorylation and dose-dependent manner. CSE-activated nuclear IKKα PIAS Ser90 phosphorylation in the IKKα-mediated nega- mediatesthepro-inflammatorygenetranscriptionthrough tiveregulationofNF-κBactivity(Figure1). phospho-acetylation of RelA/p65 and histone H3 [42], suggesting nuclear IKKα-targeted histone H3, SMRT, and NuclearfunctionsofIKKαinNF-κB-independentgene CBP/p300playaroleinCSE-inducedNF-κBactivation. transcriptionregulation In contrast to activating NF-κB in response to proin- Nuclear IKKα-mediated histone H3 phosphorylation is flammatorystimuli,IKKαkinaseactivityhasbeenreported involved in c-fos upregulation in a NF-κB-independent to be required for the termination of NF-κB activation manner in response to EGF [45] and UV [46] stimula- [43]. In response to the systemic challenge with the tion (Figure 2). Dong et al. demonstrated that Ser32 Gram-positive human pathogen group B Streptococcus phosphorylation, which is required for c-Fos protein sta- (GBS),transgenicmiceexpressingtheinactivatablevariant bility and promoter recruitment of c-Fos, requires the ofIKKα(AA)showedhigherbacterialclearancebutacce- kinase activity of nuclear IKKα [46], indicating that the lerated mortality compared with the wild-type mice. The chromatin regulation by nuclear IKKα is not limited to exacerbated inflammatory phenotype was believed to be NF-κB-targeted genes but also affects gene transcrip- associated with this paradoxical result. Indeed, after tions regulated through targeting various transcription administration of the Escherichia coli-derived LPS, factors or cofactors (Figure 2). For example, IKKα- transcripts of several pro-inflammatory and antiapoptotic mediated suppression of SMRT is required not only for NF-κB-target genes were higher in IKKαAA/AA mice than NF-κB activation [18] but also Notch-dependent tran- in the littermate controls, indicating a role of IKKα in scription [17], implying that nuclear IKKα may function terminating the activation of classical NF-κB pathway in as a common epigenetic regulator for gene transcription. response to LPS-induced Toll-like receptor (TLR) signal- Our recent study further indicated that nuclear IKKα ing. It was then demonstrated that IKKα activity is may also derepress Notch-dependent transcription by required to accelerate the removal of RelA/p65 and c-Rel diminishing the gene expression of NUMB [47], which from pro-inflammatory gene promoters and the turnover targets Notch1 for lysosomal degradation through of these NF-κB subunits by specifically phosphorylating protein-protein interaction [48]. We identified forkhead p65 within its transactivation domain at Ser536. These box protein A2 (FOXA2) as the transcription factor for events terminate LPS-induced NF-κB activation, leading NUMB gene transcription and demonstrated that IKKα to the negative regulation of macrophage activation and reduces the transcriptional activity of FOXA2 via bind- inflammation [43] (Figure 1). While much attention has ing to and phosphorylating it at Ser107 and Ser111 [47]. focused on pro-inflammatory signaling, this study Therefore, nuclear IKKα may also enhance Notch- explored an opposing but complimentary role of IKKα in dependent gene transcription by suppressing FOXA2/ resolving inflammation. Since IKKα also showed nuclear NUMBsignalingpathway(Figure2).InadditiontoNotch, function in histone phosphorylation in LPS-treated IKKα but not IKKβ was found to be involved in estrogen macrophage [32], this raises the possibility that LPS- receptor (ER)-mediated gene transcription by binding to activated IKKα also phosphorylates p65 at Ser536 in the the estrogen-responsive elements (EREs)-containing pro- nucleustoterminatethetranscriptionalactivityofNF-κB. motersand phosphorylating histone H3, ERα, or coac- However,thishypothesisseemscontradictorytotheprevi- tivators such as AIB1/SRC-3. Coordinated promoter ous finding that TNF-α-induced NIK/IKKα complex recruitmentofERsandspecificcoactivators,suchasSRC- phosphorylates p65 at Ser536 in the nucleus to enhance 1, AIB1/SRC-3, GRIP1, CBP/p300, PCAF, CARM1, and NF-κB activity [25]. These findings suggest that the tran- PRMT1, is required for estrogen-regulated transcriptional scriptional activity of NF-κB is not determined merely by activation.ByformingatranscriptionalcomplexwithERα HuangandHungJournalofBiomedicalScience2013,20:3 Page5of13 http://www.jbiomedsci.com/content/20/1/3 Figure2TherolesofnuclearIKKαintheregulationofNF-κB-independentgenetranscription.NuclearIKKαenhancesNotch-dependent genetranscriptionalbyphosphorylatingandremovingco-repressorSMRTfromtargetgenepromoters.IKKαalsocontributestoNotch transcriptionalactivitythroughphosphorylatingandinactivatingFOXA2,whichsubsequentlyleadstoNUMBsuppression.Bydirecttargeton transcriptionfactors,nuclearIKKαalsoincreasesAP-1,ERα,andE2F-mediatedgenetranscription.PhosphorylationofSRC3atSer857bynuclear IKKαalsocontributestoERαtranscriptionalactivity. and AIB/SRC3, IKKα mediates histone H3 phosphoryl- influences estrogen-mediated cell cycle progression by ation on the promoters of several estrogen-responsive modulatingE2F1atboththetranscriptionalandposttran- genes. In addition, nuclear IKKα also enhances the activ- scriptional levels [20]. Taken together, IKKα appears to ities of ERα and AIB1/SRC-3 through phosphorylation of modulate various epigenetic signaling pathways to regu- theirSer118andSer857respectively(Figure2),leadingto latespecificsetsofgenes. the increase in cyclin D1 and Myc expression and estrogen-mediated breast cancer cell growth [19]. RegulationofapoptosisbynuclearIKKα Since IKKα-activated SRC3 is also required for tran- NF-κB is activated to control apoptosis upon exposure scriptional activity of NF-κB [49], SRC-3 coactivator, to various cytotoxic stimuli, including reactive oxygen in addition to histone H3 and SMRT, may also be species (ROS). Recent evidence suggests a negative regu- another target of nuclear IKKα to increase general latory role of activated NF-κB in ROS-elicited JNK gene transcriptions. signaling to antagonize apoptosis [50]. Interestingly, the Inresponsetoestrogenstimulation,IKKαalsoregulates Src-dependent Tyr42 phosphorylation but not IKK- cell cycle progression through modulating E2F1-depedent mediated Ser32/36 phosphorylation of IκBα contributes transcription [20]. The Rb/E2F pathway controls G1/S to ROS-induced NF-κB activation in a proteolysis- phasetransitionbyactivatingexpressionofgenesrequired independent mechanism [51,52]. Although hydrogen for DNA replication. Silencing of IKKα but not IKKβ sig- peroxide stimulation has been shown to induce IKKα nificantly reduced estrogen-induced cell cycle progression activity and thus its nuclear translocation, it had no and transcription ofthe E2F1geneas well as other E2F1- effect on NF-κB activation [53], suggesting that IKKα is responsivegenes,includingthymidinekinase1,proliferat- not involved in the ROS-induced NF-κB activation. In ing cell nuclear antigen, cyclin E, and cdc25A, indicating contrast to the pro-survival role of NF-κB, nuclear IKKα thatIKKαplaysacriticalroleinregulatingE2F-dependent plays an opposite function in ROS-mediated apoptosis gene transcription. Through association with E2F1, IKKα throughmodulatingp53transcriptionalactivity(Figure3). isrecruitedtoE2F-1responsivepromotersandpotentiates In the nucleus, IKKα enhances p53-mediated GADD45 theabilityofp300/CBP-associatedfactortoacetylateE2F1 and BAD gene expressions by phosphorylating p53 at fortranscriptionalactivationinresponsetoestrogentreat- Ser20 [53] and stabilizing p53 protein levels [54], leading ment(Figure2).ThesefindingssuggestthatnuclearIKKα totheinductionofapoptosisinresponsetoROSexposure. HuangandHungJournalofBiomedicalScience2013,20:3 Page6of13 http://www.jbiomedsci.com/content/20/1/3 Figure3NuclearIKKαtargetsp53andp73tomediateapoptosis.InresponsetoDNAdamageinducedbyROSandcisplatin,nuclearIKKα stabilizesp53andp73proteinlevelrespectivelytopromoteapoptosis. In response to DNA damage, both p53 and its homolog family of transcription factors to activate the expression p73 function against NF-κB in deciding cell fate. Treat- of cyclin E and several other genes required for the cell ment withcisplatin induces IKKα nuclear translocation in cycle progression [60,61]. The direct binding of NF-κB humanosteosarcoma-derivedU2OScellsandhepatocellu- on the promoter region of cyclin D1 gene and the pro- larcarcinomaHepG2cellsinanATM-dependentmanner nounced reduction of cyclin D1 expression by NF-κB in- [23,55].AsshowninFigure3,nuclearIKKαalsostabilizes hibition provide additional evidence for the involvement p73 protein through physical interaction in response to of cyclin D1 transcription in NF-κB-mediated cell cycle cisplatin [23]. Although the exact residue of p73 phos- progression [62-64]. However, several studies showed phorylated by IKKα has not yet been identified, a study contradictory results in which NF-κB activation by over- showed that nuclear IKKα phosphorylates p73 within its expression of p65 or c-Rel causes cell-cycle arrest and N-terminal region, which may protect p73 from ubiquiti- induces cells to commit to terminal differentiation nation and proteasomal degradation [23]. These findings [65,66]. For instance, the G1-arrest by p65 occurs in suggestanindispensableroleofIKKαincisplatinsensitiv- pro-B but not in mature B cells [66], suggesting that this ity. Different from ROS-induced nuclear IKKα on p53 event depends on cell developmental stage. Interestingly, stability in ROS-treated human MOLT-4 and HL-60 mouse with IKKα gene inactivation also had an unex- leukemia cells, cisplatin-activated IKKα nuclear transloca- pected excessive proliferation of the skin basal layer due tion did not lead to p53 stabilization in HepG2 cells to the absence of epidermal differentiation [67,68]. The [53,54], suggesting that IKKα-dependent protein sta- specific roles of NF-κB in IKKα-mediated cell cycle ar- bilizationiscelltype-andstimulus-specific. rest and subsequent differentiation in keratinocyte was initially proposed based on the observation that NF-κB NuclearIKKαisessentialforcellcyclearrestand activation is not detectable in keratinocytes from IKKα- differentiationofkeratinocyteintheepidermisandthe null mouse skin [69]. However, a subsequent study morphogenesisofskeletalandcraniofacialmorphogenesis pointed out that IKKα controls epidermal keratinocyte Unlike its well-established role in anti-apoptosis, the in- differentiation independently of NF-κB activation but volvement of NF-κB in regulating cell cycle progression regulates cyclin D1 protein stability in which cyclin D1 remains unclear. NF-κB activation is required for cell is overexpressed and predominantly localized in the nu- cycling in fibroblast [56], regenerating liver cells [57], cleus of IKKα−/− MEF cells compared with parental breast cancer cells [58], and HeLa cells whereas NF-κB MEF cells [70]. In vitro binding and kinase assays inhibition impairs cell cycle progression and retardation showed that IKKα directly binds cyclin D1 and phos- of G1/S transition [59]. During cell cycle, D-type cyclins phorylates it at Thr286. The cytoplasmic expression and (cyclin D1, D2, and D3) are critical for G1 to S phase increased degradation of cyclin D1 by reconstitution of progression. By phosphorylating the retinoblastoma IKKα in knockout cells further suggest that this phos- tumor suppressor protein (pRb), cyclin D with its part- phorylation by IKKα is required for nuclear export and ner cyclin-dependent kinases (CDKs), releases the E2F turnover of cyclin D1 [71]. The predominantly nuclear HuangandHungJournalofBiomedicalScience2013,20:3 Page7of13 http://www.jbiomedsci.com/content/20/1/3 localizedcyclinD1 implies that IKKα mayphosphorylate craniofacialmorphogenesis [70,74],whichisnot observed cyclinD1inthenucleusandregulatesitsnuclearexport. in mice with systemic inhibition of NF-κB [75]. The The potential nuclear function of IKKα in facilitating results from these studies further support the dispensable cyclin D1 protein degradation but not in NF-κB activa- role of NF-κB in nuclear IKKα-mediated keratinocyte dif- tion may be attributed to IKKα-mediated cell cycle ar- ferentiation. By introducing an epidermal-specific IKKα rest and differentiation of keratinocytes (Figure 4). transgene into IKKα-deficient mice, most of these Indeed, keratinocyte differentiation is associated with morphological abnormalities were completely rescued, increased nuclear distribution of IKKα. Inactivation of suggestingthat nuclear IKKα-dependent epidermal differ- the NLS by site-directed mutagenesis prevents IKKα entiation control skeletal and craniofacial morphogenesis from entering the nucleus without affecting its kinase [8].InadditiontotargetingcyclinD1proteindegradation, activity and blocks the IKKα-induced differentiation of another potential mechanism by which nuclear IKKα primary cultured IKKα−/− keratinocyte, supporting an affects keratinocyte differentiation and craniofacial and essential role of nuclear IKKα in the keratinocyte differ- skeletalmorphogenesis isthrough repression ofthe fibro- entiation [8]. Likewise, Marinari et al. also found that blastgrowthfactor(FGF)familymembers[8],whichbind nuclear IKKα can act as a tumor suppressor in stratified toFGF receptor (FGFR) toantagonizebonemorphogenic epithelia [72]. After stimulation with TGFβ, IKKα accu- protein (BMP) signaling [76]. Since reintroduction of a mulates in the nucleus of keratinocytes and occupies the catalyticallyinactiveformofIKKαinIKKα−/−miceisstill promoter of genes responsive to TGFβ-SMAD signaling able to rescue epidermal differentiation and skeletal mor- to mediateTGFβ-induced Ovol1 and Mad1 upregulation phogenesis, the developmental functions of IKKα have and Myc downregulation (Figure 4). Such activity of nu- been proposed to be independent of its protein kinase clear IKKα is important for the anti-proliferative TGFβ activity[76].Therefore,nuclearIKKαmayalsocontribute pathway. In contrast, the expression and nuclear loca- tothesuppressionofFGF transcriptionthroughakinase- lization of IKKα are gradually reduced during malignant independent manner, hence excluding its involvement in progression of squamous cell carcinoma (SCC) and ac- phosphorylating histone H3 (Figure 4). Exploration of the quisition of an invasive phenotype [72], which supports kinase-independent roles of nuclear IKKα awaits further the tumor suppressive role of nuclear IKKα. However, studies. the function of TGFβ-induced nuclear IKKα seems to In addition to G1/S transition, IKKα also has a role in counter its metastatic role in breast cancer cells [73] regulatingtheMphaseofcellcycleasshowninFigure4. (pleaseseebelow),suggestingakeratinocyte-specificrole Progression through the M phase of cell cycle is ofnuclear IKKαinsuppressingcellproliferation. dependent on several mitotic kinases, including those of Besides the failure of epidermal differentiation, IKKα- the Aurora families [77]. Aurora A localizes to the deficient mice also exhibit abnormalities in skeletal and centrosome and functions in centrosome maturation Figure4RegulationsofcellcycleprogressionbynuclearIKKα.Inthenucleus,IKKαisinvolvedincellcyclearrestatG1/Stransitionby increasingSmadtranscriptionalactivity,facilitatingcyclinD1proteasomaldegradation,andFGFgeneexpression.NuclearIKKαalsopromotesG2/ MphaseprogressionbyincreasingkinaseactivityofAuroraAandbyde-repressing14-3-3σgeneexpressionthroughpreventingDNAand histonemethylationonthepromoter. HuangandHungJournalofBiomedicalScience2013,20:3 Page8of13 http://www.jbiomedsci.com/content/20/1/3 and separation [77], and knock down of Aurora kinase NuclearfunctionofIKKαintumorigenesisandmetastasis A by siRNA increased the percentage of mitotic cells Constitutive activation of NF-κB has been found in with high levels of Plk1 and cyclin B1 [78]. In a similar many types of tumor cells. Most of these studies report pattern to Aurora A siRNA knockdown, Prajapati et al. an increased IKK activity that results in phosphorylation showed that silencing of IKKα but not IKKβ by siRNA of IκBα; however, some have found little or no changes also increased the number of HeLa cells at the G2/M in the subcellular localization of p65 in some of the phase and the levels of Plk1 and cyclin B1 [79]. These tumor cells. For instance, Fernández-Majada et al. results further revealed that IKKα is associated with reported that the increased IKK activity in colorectal Aurora A in the centrosome and directly phosphorylates cancer (CRC) cell lines and primary CRC is concomitant Aurora A at Thr288 [79], suggesting a nuclear function with undetectable levels of nuclear p65 and p52, which of IKKα in regulating the M phase of the cell cycle is consistent with the absence of p65 and p52 on differ- through Aurora A phosphorylation. In addition to tar- ent promoters of NFκB-target genes detected by ChIP geting Aurora A, chromatin-bound IKKα also maintains analysis. Theseresults indicatethat NFκB activation may the progression of G2/M phase during the cell cycle by not be the main consequence of IKK activity in colorec- preventing the silencing of 14-3-3σ, a check point tal tumors, reflecting the substrate specificity of different protein for G2/M phase transition [80]. In IKKα-defi- IKK complexes. By immunohistochemistry staining and cient keratinocytes that showed cell cycle arrest at the subcellular fractionation followed by Western blot ana- G2/M phase, the SUV39h1 histone trimethyltransfer- lysis, they also showed that IKKα is present in the nu- ase and the Dnmt3a DNA methyltransferase were cleus of most primary colorectal tumor tissues and CRC found to associate and methylate histone H3 lysine-9 cell lines but not in HS27 or HEK-293 control cells. The (K9) and 14-3-3σ locus DNA, respectively, which then increase in nuclear IKK activity in colorectal tumors is silenced 14-3-3σ expression. Reintroduction of wild- significantly correlated with SMRT phosphorylation at type (WT) IKKα, but not its chromatin-unbound Ser2410 and its cytoplasmic translocation (Figure 5). At mutants bearing defects within the leucine zipper do- the chromatin level, the association of IKKα to specific main and helix-loop-helix motif, restored the 14-3-3σ Notch target promoters results in the release of expression by preventing the association of SUV39h1 chromatin-bound SMRT and thus activating hes1, hes5, and Dnmt3a with the 14-3-3σ locus, indicating that or herp2/hrt1 transcription, which promotes cell prolif- chromatin-associated IKKα prevents 14-3-3σ from eration by repressing transcription of the cyclin- hypermethylation (Figure 4). Interestingly, the kinase dependent kinase inhibitor p27Kip1 [17]. In addition, we activity of IKKα is dispensable for blocking 14-3-3σ reported that enhancement of Notch transactivation by hypermethylation [80], suggesting that nuclear IKKα may IKKα through inhibition of FOXA2/NUMB signaling is protect 14-3-3σ from hypermethylation through an unex- also likely to contribute to inflammation-mediated liver ploredkinase-independentmechanism. cancer progression [47]. Our other study also indicated Figure5NuclearIKKαandtumorprogression.NuclearIKKαpromotestumorgrowthbyenhancingNF-κB-andNotch-dependentgene transcriptionsandsuppressingFOXA2-mediatedgeneexpression.BypromotingSmadandSTAT3transcriptionalactivityandsuppressingmaspin geneexpression,nuclearIKKαcontributestocancermetastasis. HuangandHungJournalofBiomedicalScience2013,20:3 Page9of13 http://www.jbiomedsci.com/content/20/1/3 thatconstitutivelyactivatedIKKα,foundincertainhuman Lys236,andLys237withinthekinasedomainofIKKαhas cancers,includinglung,liver,pancreatic,andovariancan- been shown to contain the NLS. Mutation of these resi- cers, can phosphorylate and direct CBP to bind preferen- dues attenuated the spontaneous nuclear import of IKKα tiallytoNF-κB butnot p53,therebyfavoringproliferation but did not interfere with its kinase activity or binding to andsurvivaloverp53-dependentapoptosis[15]. IKKγ [8]. We also characterized the signaling peptide for The epithelial to mesenchymal transition (EMT) is a IKKα nucleo-cytoplasmic shuttling in response to HBx crucial step in tumor progression in many tumor types. overexpression and found that in addition to these three IndependentlyofNF-κBactivation,nuclearIKKαhasbeen lysines, two additional lysines (233 and 240) are also implicated in EMT by enhancing gene expression of required for the nuclear translocation of IKKα [14]. The SNAIL and SLUG transcription factors to downregulate energy for the importin-based nuclear transport is expression of the adherens junction protein E-cadherin. provided by the small Ras family GTPase, Ran [85]. A As shown in Figure 5, IKKα enters the nucleus and regu- dominantnegativemutantofRanhasbeenreportedtoin- lates gene expression of SNAIL and SLUG by interacting hibit IKKα nuclear translocation [25], suggesting that the with SMAD3 and controlling SMAD complex formation nuclear import of IKKα requires importins. However, the on the promoters of these two transcription factors in specific molecules that are involved in NLS-mediated response to TGFβ activation, leading to metastasis of IKKαnucleartranslocationremaintobeinvestigated. breast cancer cells [73]. Tumor-infiltrating immune cells Likewise, nuclear export signals (NES), which are expressinglymphotoxin-β[81]andRANKL[24]havealso recognized by a soluble export receptor (also known as been found to induce activation and nuclear localization Exportin 1 or CRM1), mediate nuclear export [86]. A ofIKKαinprostaticepithelialtumorcells(Figure5).After study in 2002 by Birbach et al. showed that presence castration,activatedSTAT3hasbeenreportedtopromote andincubationofLMB,aninhibitorofCRM1,enhanced the transcriptional activity of unliganded androgen recep- the levels of IKKα in the nucleus [13]. This raises the tor in prostate cancer cells [82]. Lymphotoxin β-induced possibility that IKKα can shuttle out of the nucleus nuclear IKKα, in conjunction with STAT3, contributes to through the CRM1 pathway and contains an NES to the emergence of castration resistance and enhances allow for the recognition and binding of CRM1 receptor. hormone-free survival and metastasis of prostate cancer Based on the consensus NES sequence, which typically by an NF-κB-independent, cell autonomous mechanism has a leucine-rich consensus sequence in the form of [81]. By targeting histone H3 Ser10 on the promoter of LX LX LXL (L=leucine and X=any amino acid) [86], 1-3 2-3 maspin, nuclear IKKα was also proposed to mediate the there are two putative NESs (Leu601 ~ Leu612 and repression of maspin, a critical suppressor of metastasis, Leu714~Leu724) located at the C-terminus of IKKα. through an unidentified mechanism, which then commits Leucine or isoleucine substitution within the motif con- malignant prostatic epithelial cells to a metastatic fate taining residues 714–724 enhanced nuclear accumula- [24]. Similarly, we found that overexpression of HBx tion ofIKKα,thereby supporting thepresenceofan NES reducedmaspinexpressioninHep3Bcells,andexpression forIKKαnuclear export [14]. of wild-type IKKα but not its NLS mutant suppressed SinceIKKαcanenterthenucleusinresponsetodiverse maspin expression in Hep3B cells, indicating that nuclear stimuli, includingTNF-α [12,16], Helicobacter pylori [13], IKKα likely plays a role in HBx-mediated cell migration estrogen [20], EGF [45], and cisplatin [55], it is likely that and invasion via suppressing maspin expression [14]. signaling pathways, in addition to NLS and NES, are also Although nuclear IKKα has been proposed to suppress critical for regulating IKKα nucleo-cytoplasmic shuttling maspin expression via histone H3 Ser10 phosphorylation (Figure 6). A kinase-dead mutant of IKKα (IKKα-K44M) [24] (Figure 5), it is still unclear how this histone phos- has been shown to have lower nuclear accumulation than phorylation reduces the promoter activity of maspin. the wild type form, indicating that kinase activation is Taken together, these findings indicate that a specific set required for IKKα to translocate into the nucleus [13]. of genes regulated by nuclear IKKα plays a critical role in Activation of IKK complex usually involves trans- tumorigenesis and metastasis. The detailed molecular autophosphorylation by the catalytic domains of IKKα mechanismsawaitfurtherinvestigations. and IKKβ. However, knockdown of IKKβ by siRNA had noeffectonH.pylori-inducedIKKαnucleartranslocation PotentialmechanismsofIKKαnucleartranslocation [22]. This suggests that IKKα can bypass the classic IKK Exploration of various nuclear IKKα functions raised a complexactivationpathwaytoenterthenucleus.AsIKKβ fundamental question of how IKKα travels from the does not exist in the nucleus, the distinct mechanisms by cytoplasm to the nucleus. It is believed that, for a major- which the kinases are regulated may have a role in ity of proteins, NLS-bearing molecules are transported controlling the nuclear translocation of IKKα. Indeed, intothe nucleus by forminga complex with importin α/β Akt, a mitogen-activated survival factor, has been shown [83] orimportin β alone[84].A lysine-richmotif,Lys235, to increase the activity of IKKα but not IKKβ by HuangandHungJournalofBiomedicalScience2013,20:3 Page10of13 http://www.jbiomedsci.com/content/20/1/3 Figure6MolecularmechanismsofIKKαnucleartransportation.RanGTPaseactivityisrequiredforthenucleartransportofIKKαthrough interactingwithimportin-α.InresponsetoHBxoverexpressionandcisplatintreatment,phosphorylationsofIKKαatThr23andSer473byAktand ATMrespectivelypromoteitsnucleartranslocation.TheubiquitinationofIKKαisessentialfortheAkt-regulatedIKKαnuclearimport.Under exposuretoROS,activatedPKCδalsoenhancesthenuclearaccumulationofIKKα. phosphorylating it at Thr23 in response to TNF-α [87]. Conclusion Intriguingly, the signals that stimulate IKKα nuclear im- Since the first observation of nuclear localization of port, including HBx, EGF, HER2, and TNF-α, also com- IKKα more than a decade ago, the field has gained monly induce Akt activation. Akt-enhanced nuclear tremendousinsightintothedistinctregulationandfunc- expression of IKKα is further augmented by overexpres- tions of nuclear IKKα. Other than IκB protein in the sion of ubiquitin, suggesting that ubiquitination plays a cytoplasm, these studies added histone and transcrip- role in Akt-regulated IKKα nuclear transportation [14]. tionalco-factors asnucleartargetsofIKKαforactivation Further investigations are necessary to identify the E3 of NF-κB-dependent transcription. By targeting a grow- ligaseandtheubiquitinationsitesofIKKα. ing list of substrates in the nucleus, IKKα has also been In response to cisplatin-induced DNA damage, ATM implicated in a variety of biological functions, including has been shown to activate and phosphorylate IKKα at apoptosis, tumor suppression, immune functions, cell Ser473 in an in vitro kinase assay. Treatment with ATM proliferation, and chromatin remodeling in an NF-κB- inhibitors blocked the nuclear IKKα accumulation by independent manner. Dysregulation of nuclear IKKα has cisplatin,suggestingthatATMplaysaroleinthenuclear been further linked to diabetes [88]. Contextual condi- translocation of IKKα. In addition, the active form of tioned fear memory may also transduce IKKα to the nu- ATM was shown to colocalize with IKKα in the nucleus cleus of hippocampus for transcriptional regulation after to mediate cisplatin-induced p73 protein stabilization memory recall [89]. These findings uncovered functional and apoptosis [55]. These findings suggest that Ser473 diversity of nuclear IKKα and other probable roles phosphorylation by ATM may be a critical posttransla- worthy of further exploration. For example, the involve- tional modification for IKKα nuclear import and func- ment of nuclear IKKα in the termination of NF-κB sig- tions in response to cisplatin treatment. Similarly, in naling is an attractive yet under-developed area. The response to ROS exposure, PKCδ has also been demon- exploration of the nuclear role of IKKα in terminating strated to increase the kinase activity and nuclear trans- NF-κB activity could lead us to understand how inflam- location of IKKα through protein-protein interaction mation is resolved. The kinase-independent function of (Figure 6). PKC-activated nuclear IKKα promotes the nuclear IKKα, which has been shown to control FGF stability of p53 protein and mediates ROS-induced suppression during epidermal differentiation and skeletal apoptosis [53]. However, the phosphorylation site morphogenesis, is also another interesting area requiring mediated by ROS-activated PKC remains unclear. It further investigations. Addressing the kinase-independent would be of interest to further address whether phos- functionsofIKKαwilllikelyprovidemore comprehensive phorylation of IKKα by Akt, ATM, or PKCδ at different explanation for the distinct roles between IKKα and its residuesaffectsitssubstrate preference inthenucleus. homologue,IKKβ.