15. Monsivais, P., Clark, B.A., Roth, A., andHausser, M. (2005). Determinants ofaction potential propagation in cerebellarPurkinje cell axons. J. Neurosci. 25,464–472.

16. Borst, J.G., and Sakmann, B. (1999).Effect of changes in action potentialshape on calcium currents and transmitterrelease in a calyx-type synapse ofthe rat auditory brainstem. Philos.Trans. R. Soc. Lond. B 354,347–355.

17. Nicholls, J., and Wallace, B.G. (1978).Modulation of transmission at an

inhibitory synapse in the central nervoussystem of the leech. J. Physiol. 281,157–170.

18. Shimahara, T., and Tauc, L. (1975).Multiple interneuronal afferents to thegiant cells in Aplysia. J. Physiol. 247,299–319.

19. Shapiro, E., Castellucci, V.F., andKandel, E.R. (1980). Presynapticmembrane potential affects transmitterrelease in an identified neuron in Aplysiaby modulating the Ca2+ and K+ currents.Proc. Natl. Acad. Sci. USA 77,629–633.

20. Debanne, D. (2004). Informationprocessing in the axon. Nat. Rev.Neurosci. 5, 304–316.

Wolfson Institute for BiomedicalResearch and Department ofPhysiology, University College London,Gower Street, London WC1E 6BT, UK.E-mail: m.hausser@ucl.ac.uk

DOI: 10.1016/j.cub.2006.07.007

Current Biology Vol 16 No 15R588

IKKe Signaling: Not Just NF-kB

IkB kinases (IKKs) are key components of NF-kB signaling pathways ininnate immunity and inflammation. Surprisingly, three recent reportsimplicate IKKs in Drosophila in seemingly unrelated functions, includingnon-apoptotic caspase activation and cytoskeleton organization.

Andreas Bergmann

NF-kB transcription factors arekept inactive by cytoplasmicsequestration through complexformation with inhibitory IkBproteins [1]. NF-kB stimulation – forexample initiated by Toll-likereceptors in innate immunity orin response to proinflammatorycytokines such as TNFa – requiresdissociation of the NF-kB/IkBcomplex. IkB kinases (IKKs)were initially identified asa high-molecular weight complexcapable of site-specificphosphorylation of IkB-a [2]. Thisphosphorylation triggersubiquitin-mediated degradation ofIkB-a, and the release of NF-kBtranscription factors, whichtranslocate into the nucleus [2].Subsequent analysis identified twocatalytic subunits (IKKa and IKKb)and a structural component of thiscomplex (IKKg/NEMO). While theIKKa/b/g complex is required forNF-kB activation in response tomost NF-kB inducers, the role oftwo related kinases known asIKKe/IKKi and TBK1/NAK/T2K isless clear.

In Drosophila, two independentimmune signaling pathwayscontrol the activity of distinctNF-kB-like proteins [3]. Whilethe Toll/anti-fungal pathwayrequires Dorsal and Dif, theIMD/anti-bacterial pathway leadsto activation of Relish [3]. Dorsaland Dif are rendered cytoplasmic

in complex with the only IkB-likeprotein, termed Cactus.Phosphorylation of Cactus isrequired for its degradation [4–6],but the responsible kinase hasnot been identified. TheDrosophila genome encodes twoIKK genes. DmIKKb (or DLAK) ismost similar to human IKKb, andis involved in Relish activation[7]. That leaves the secondDrosophila IKK, DmIKKe (alsoknown as Ik2), as a candidate forthe Cactus kinase. However,recent reports [8,9], includingone in this issue of CurrentBiology [10], rule out a function ofDmIKKe as Cactus kinase.Instead, DmIKKe modulatescaspases for a non-apoptoticfunction and controls bothactin and microtubulecytoskeletons.

DmIKKe as a Negative Regulatorof Diap1 Protein StabilityAs in vertebrates, apoptosis inDrosophila is triggered byactivation of caspases, a highlyspecialized class of cell deathproteases. In surviving cells,caspases are kept inactive throughcomplex formation with inhibitorof apoptosis proteins (IAPs),most notably Drosophila IAP1(Diap1) [11]. In response tocell death-inducing signals,pro-apoptotic proteins such asReaper stimulate the ubiquitylationand degradation of Diap1,releasing caspases from IAP

inhibition and triggering apoptosis[11]. Interestingly, the recent paperby Kuranaga et al. [9] identifiesmutations in DmIKKe

as dominant suppressors ofReaper-induced cell death [9].Subsequent analysis showed thatloss of DmIKKe increases thestability of the Diap1 protein,providing an explanation forthe observed suppression ofReaper-induced apoptosis [9].These observations suggest thatwild-type DmIKKe destabilizesDiap1, leading to Caspaseactivation, a conclusion whichwas confirmed in cell cultureexperiments and in transgenicflies.

Destabilization of Diap1 appearsto be the result ofphosphorylation by DmIKKe.Interestingly, human TBK1/NAK/T2K was able to promotephosphorylation and degradationof human XIAP [9], suggestingconservation ofIKKe-mediated control of IAPstability. DmIKKe-mediated Diap1degradation is independent ofReaper. Overexpression ofDmIKKe in cell death deficient(i.e. reaper mutant) backgroundstill induced Diap1 instability andapoptosis [9]. This is a strikingfinding, as it suggests that controlof Diap1 stability and thus caspaseactivation in Drosophila occursthrough distinct pathways,including the classical apoptoticpathway and as well as by IKKe

signaling.

DmIKKe Controls Diap1 in aNon-Apoptotic SettingDespite the fact thatoverexpression of DmIKKe inducesa strong apoptotic phenotype,developmental cell death appears

DispatchR589

to be unaffected in DmIKKe

mutants or in response toinactivation by RNAi [9]. However,these studies need to bereinvestigated as the RNAiapproach used can often causehypomorphic effects, and thematernal contribution was notremoved for the embryonicanalysis of DmIKKe mutants.Nevertheless, under normaldevelopmental conditions,DmIKKe seems to control Diap1protein levels and thus caspaseactivity not for the purpose ofapoptosis induction, butinstead in a much more subtle wayfor a non-apoptotic function ofcaspases. This conclusion wassubstantiated using a sensitivecaspase reporter whichdemonstrated that DmIKKe

modulates caspase activity onlymildly [9]. Non-apoptoticfunctions of caspases have beenreported previously, includingsperm individualization [12,13],border cell migration [14], neuralstem cell differentiation [15],erythrocyte, keratinocyte andlens differentiation, as well asT-cell and B-cell proliferation [16].However, conceptually it is stilldifficult to conceive how caspaseactivation in some settingsinduces apoptosis, while inothers it does not. The report byKuranaga et al. [9] suggeststhat DmIKKe may provide acheckpoint for apoptoticversus non-apoptotic activity ofcaspases.

DmIKKe Regulates the ActinCytoskeletonWhich non-apoptotic cellularprocess is influenced by DmIKKe

and Diap1? Clues to answer thisquestion came from the secondstudy published in this issueof Current Biology. Oshimaet al. [10] identified DmIKKe ina misexpression screen forregulators of epithelialmorphogenesis of the trachealsystem. Overexpression ofDmIKKe in the tracheal systemdoes not induce apoptosis, butinstead disrupts F-actin dynamicsresulting in loss of epithelialintegrity. A convenient model tostudy actin dynamics are bristleswhich are constructed with rings ofmembrane-attached, cross-linked

actin bundles. Consistently, theloss-of-function phenotype ofDmIKKe mutants in sensorybristles resembles the phenotypeof actin-regulatory mutants(Profilin, b-subunit of cappingprotein) or of bristles treated withF-actin inhibitors, suggesting thatDmIKKe regulates F-actinassembly [10]. Dominant negativeDmIKKe also enhanced theoverexpression phenotype ofProfilin, further supporting thenotion that DmIKKe regulatesF-actin dynamics [10]. In addition,ectopic sites of actinpolymerization in the ooplasm ofDmIKKe mutants were observed[8]. In conclusion, the actincytoskeleton is disrupted inDmIKKe mutants.

The link to Diap1 in this studywas established by analyzing theantennal arista phenotype. RNAiinactivation of DmIKKe causesexcessive branching of the arista[10] as has also been observedin hid mutants [17], anotherpro-apoptotic gene similar toreaper [11]. Interestingly, diap1mutants display the opposite, i.e.branchless or thread, phenotype ofthe arista [17]. Theseobservations suggest thatDmIKKe and Diap1 haveopposing functions for aristamorphogenesis. Consistently,the excessive branchingphenotype of dominant negativeDmIKKe was suppressed byinactivation of Diap1 andenhanced by overexpression ofDiap1, confirming the negativerelationship between DmIKKe

and Diap1 [10]. Diap1 haspreviously been shown topromote border cell migrationin an apparently non-apoptoticfunction through stimulationof actin polymerization [14]. Inline with this, overexpressionof DmIKKe prevents border cellmigration without inducingapoptosis [10], furthersuggesting that IKKe throughits effect on Diap1 couldresult in decreased actinpolymerization.

Interestingly, reduction of theactivity of Dronc, an initiatorcaspase controlled by Diap1,enhanced the antennal aristaphenotype caused by DmIKKe,while inhibition of effector

caspases had no effect on aristamorphology [10]. Theseobservations imply that thenon-apoptopic activity of Diap1 ismediated by the initiator caspaseDronc, but not by apoptoticeffector caspases. Somehow, ina manner that is not wellunderstood, DmIKKe modulatesthe activity of Dronc via Diap1, suchthat Dronc regulates F-actinpolymerization in a non-apoptoticmanner.

DmIKKe Is Not the Cactus KinaseBut Is Involved in mRNALocalizationThe third report by Shapiro andAnderson [8] attempted toidentify DmIKKe as the Cactuskinase. Cactus is the only IkBprotein in Drosophila and itsphosphorylation is required fordegradation [4–6]. However, thekinase responsible has not beenidentified. Because DmIKKb is notinvolved in Cactus inactivation (seebelow), DmIKKe was a goodcandidate. In addition to innateimmunity, Cactus also controlsdorsoventral polarity of theDrosophila embryo, and mutationsin the Cactus kinase would beexpected to cause a dorsalizedphenotype [8]. However, DmIKKe

mutations cause the oppositeventralized phenotype, ruling outDmIKKe as the Cactus kinase. Inaddition, embryos fromhomozygous DmIKKe mothersshow a bicaudal phenotype, i.e.a lack of anterior structures anda duplication of posterior ones [8].This combination of ventralizedand bicaudal phenotypes isindicative of abnormal mRNAlocalization in the developingoocyte. During oogenesis, thecorrect localization of a numberof mRNAs is critical for thedevelopment of theembryo. For example, duringmid-oogenesis oskar mRNAlocalizes to the posterior pole, andgurken mRNA to the anterior dorsalcorner of the developing oocyte.In DmIKKe mutant oocytes, oskarmRNA is localized at both theanterior and posterior poles, thuscausing the bicaudal phenotype.Although gurken mRNA waslocalized to the anterior marginof the oocyte, it was notconcentrated dorsally, thus

Current Biology Vol 16 No 15R590

explaining the ventralizedphenotype [8]. These findingssuggest that DmIKKe is required forthe localization of specific mRNAsduring oogenesis.

Correct localization of thesemRNAs depends on microtubulesand microtubule motors, mostnotably dynein and kinesin.Subsequent analysis showed thatmRNA mis-localization in DmIKKe

mutant oocytes is associated withdefects in dynein-mediated,minus-end-directed transportprocesses during mid-oogenesis[8]. Kinesin function is alsodisturbed in DmIKKe oocytes;however, this appears to besecondary to dynein disruption[8]. Therefore, the abnormalmRNA localization in DmIKKe

mutant oocytes can be attributedto defects in organization ofmicrotubule minus-ends givingrise to ventralized and bicaudalphenotypes of DmIKKe mutantembryos [8].

Intersection between IKK andCaspase Pathways in DrosophilaThe reports discussed abovedemonstrate that DmIKKe linksthe NF-kB and caspase pathwaysin a non-apoptotic manner. Thisis already the second exampleof such an intersection. TheDrosophila caspase Dredd,a caspase-8 homolog, is notinvolved in apoptosis, but insteadin the activation of the NF-kB-likeprotein Relish [7]. Relish isproduced as a p100/p105NF-kB-like precursor protein thatneeds to be cleaved for activation.In response to bacterial infection,DmIKKb, the other DrosophilaIKK, phosphorylates Relish [18].Phosphorylated Relish is then thetarget of proteolytic cleavage byDredd for activation [7]. It thusappears that during evolution IKKshave recruited various caspases(Dredd, Dronc) for non-apoptoticfunctions.

Future DirectionsAll of the mentioned studiesrevealed that DmIKKe governsbasic aspects of both actincytoskeleton and microtubuleorganization. The control of actinpolymerization appears to bemediated by a non-apoptoticfunction of Diap1 and the caspase

Dronc. It will be exciting todetermine whether Diap1 andDronc also control microtubuleorganization, or whether this isa distinct function of DmIKKe. Inthis context, it is interesting to notethat mutations in spn-F, whichencodes a novel protein, causeexactly the same phenotype asDmIKKe [19]. Spn-F associateswith microtubule minus-ends, andDmIKKe and Spn-F have beenshown to interact in yeast. Thus,Spn-F may be a regulatory targetof DmIKKe in microtubuleorganization.

IKK proteins are importantcomponents of NF-kB signaling.Although a role of DmIKKe asCactus kinase in dorsoventralpatterning of the embryo has beenexcluded, none of the discussedreports — surprisingly — actuallyhas addressed whether DmIKKe

has a role in the immune responsein Drosophila. This is still an openquestion. The genetic studies inDrosophila discussed here providestrong evidence for anNF-kB-independent functionof IKKs, most notable IKKe.The observation that humanTBK1/NAK/T2K controls proteinlevels of XIAP suggest that theNF-kB-independent function isconserved. Future work willdetermine whether the mammalianproteins control actin andmicrotubule dynamics in a similarmanner as their Drosophilacounterpart.

References1. Silverman, N., and Maniatis, T. (2001).

NF-kappaB signaling pathways inmammalian and insect innateimmunity. Genes Dev. 15,2321–2342.

2. Chen, Z.J., Parent, L., and Maniatis, T.(1996). Site-specific phosphorylationof IkappaBalpha by a novelubiquitination-dependent proteinkinase activity. Cell 84,853–862.

3. Royet, J., Reichhart, J.M., andHoffmann, J.A. (2005). Sensing andsignaling during infection in Drosophila.Curr. Opin. Immunol. 17, 11–17.

4. Bergmann, A., Stein, D., Geisler, R.,Hagenmaier, S., Schmid, B.,Fernandez, N., Schnell, B., and Nusslein-Volhard, C. (1996). A gradient ofcytoplasmic Cactus degradationestablishes the nuclear localizationgradient of the dorsal morphogen inDrosophila. Mech. Dev. 60,109–123.

5. Fernandez, N.Q., Grosshans, J.,Goltz, J.S., and Stein, D. (2001). Separableand redundant regulatory determinants inCactus mediate its dorsal groupdependent degradation. Development128, 2963–2974.

6. Reach, M., Galindo, R.L., Towb, P.,Allen, J.L., Karin, M., andWasserman, S.A. (1996). A gradientof cactus protein degradationestablishes dorsoventral polarity in theDrosophila embryo. Dev. Biol. 180,353–364.

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8. Shapiro, R.S., and Anderson, K.V. (2006).Drosophila Ik2, a member of the I kappa Bkinase family, is required for mRNAlocalization during oogenesis.Development 133, 1467–1475.

9. Kuranaga, E., Kanuka, H., Tonoki, A.,Takemoto, K., Tomioka, T.,Kobayashi, M., Hayashi, S., and Miura, M.(2006). Drosophila IKK-related Kinaseregulates non-apoptotic function ofcaspases via degradation of IAPs. Cell,in press.

10. Oshima, K., Takeda, M., Kuranaga, E.,Ueda, R., Aigaki, T., Miura, M., andHayashi, S. (2006). IKKe regulatesF-actin assembly and interactswith Drosophila IAP1 in cellularmorphogenesis. Curr. Biol. 16,1531–1537.

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13. Huh, J.R., Vernooy, S.Y., Yu, H., Yan, N.,Shi, Y., Guo, M., and Hay, B.A. (2004).Multiple apoptotic caspase cascades arerequired in nonapoptotic roles forDrosophila spermatid individualization.PLoS Biol. 2, E15.

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18. Silverman, N., Zhou, R., Stoven, S.,Pandey, N., Hultmark, D., and Maniatis, T.(2000). A Drosophila IkappaB kinasecomplex required for Relish cleavage andantibacterial immunity. Genes Dev. 14,2461–2471.

19. Abdu, U., Bar, D., and Schupbach, T.(2006). spn-F encodes a novel protein thataffects oocyte patterning and bristlemorphology in Drosophila. Development133, 1477–1484.

The University of Texas MD AndersonCancer Center, Department ofBiochemistry & Molecular Biology, 1515Holcombe Blvd – Unit 1000, Houston,Texas 77030, USA.E-mail: abergman@mdanderson.org

DOI: 10.1016/j.cub.2006.07.010