InteractiveFly: GeneBrief

I-kappaB kinase epsilon : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Caspase activation has been extensively studied in the context of apoptosis. However, caspases also control other cellular functions, although the mechanisms regulating caspases in nonapoptotic contexts remain obscure. Drosophila IAP1 (DIAP1) is an endogenous caspase inhibitor that is crucial for regulating cell death during development. Drosophila IKK-related kinase (DmIKKε; FlyBase name, IkappaB kinase-like 2) as a regulator of caspase activation in a nonapoptotic context. DmIKKε promotes degradation of DIAP1 through direct phosphorylation. Knockdown of DmIKKε in the proneural clusters of the wing imaginal disc , in which nonapoptotic caspase activity is required for proper sensory organ precursor (SOP) development, stabilizes endogenous DIAP1 and affects Drosophila SOP development. These results demonstrate that DmIKKε is a determinant of DIAP1 protein levels and that it establishes the threshold of activity required for the execution of nonapoptotic caspase functions (Kuranaga, 2006).

Inhibitor of apoptosis proteins (IAPs), originally found in baculoviruses, are present in organisms from viruses to yeasts to humans. The IAP family is comprised of endogenous caspase inhibitors that play crucial roles in developmental cell death in Drosophila and in life/death decisions in mammalian cells in various cancers. In mammals, additional roles for IAPs have been proposed in a variety of cellular processes, including the control of cell division, and in a number of different signaling cascades, such as transforming growth factor β activation, c-Jun N-terminal kinase regulation, and nuclear factor κB (NF-κB) activation. However, the role of mammalian IAPs in vivo is unclear because a loss-of-function experiment of one IAP, X-linked IAP (XIAP), showed no obvious physiological or histological defects, and the animals had normal life spans because of redundant functions of c-IAP1 and c-IAP2 (Kuranaga, 2006).

During early development, Drosophila IAP 1 (DIAP1) is an essential protein whose depletion leads to massive cell death in the embryo and the subsequent death of the fly. DIAP1 inhibits caspases through direct binding or degradation, as in the case of the initiator caspase DRONC. The stabilization of DRONC upon the degradation of DIAP1 promotes cell death. Despite the importance of DIAP1 in preventing cell death, the DIAP1 protein itself is unstable, with an in vivo half-life of approximately 30 min, suggesting that quantitative control of the DIAP1 protein level might be required to regulate caspase activation (Kuranaga, 2006).

In addition to the control of DIAP1 degradation-mediated caspase activation during apoptosis, the importance of other regulatory mechanisms of caspase activation under nonapoptotic conditions is widely recognized. Caspase activity is required not only for cell death but also for various physiological functions, including sperm individualization (Arama, 2003; Huh, 2004); neural stem cell differentiation (Fernando, 2005); erythrocyte, keratinocyte, and lens differentiation; and T cell and B cell proliferation (reviewed in Schwerk, 2003). However, the caspase-activating mechanisms of nonapoptotic conditions are largely unknown (Kuranaga, 2006).

This study has identified a novel regulator of caspase activation that promotes DIAP1 phosphorylation and degradation, using a dominant-modifier screen in Drosophila. Drosophila IKK-related kinase (DmIKKε), a homolog of the noncanonical members of IκB kinase family (IKKε/IKKι or NAK/T2K/TBK1) that regulate NF-κB activation or interferon regulatory factor (IRF) 3 and 7 activation in mammals, determines the level of DIAP1. Ectopic expression of DmIKKε causes DIAP1 phosphorylation and degradation, resulting in cell death. The mammalian homolog of DmIKKε, NAK/T2K/TBK1, also degrades and phosphorylates mammalian XIAP. Knockdown of DmIKKε in the proneural clusters of the wing imaginal disc, in which nonapoptotic caspase activity is required for proper sensory organ development, stabilizes the endogenous DIAP1 and affects Drosophila sensory organ precursor (SOP) development. These results demonstrate that DmIKKε is a determinant of the DIAP1 protein level and that it provides the threshold of caspase activity required for the execution of nonapoptotic functions (Kuranaga, 2006).

The work reported in this study identifies Drosophila IKK-related kinase as a regulator of DIAP1 turnover and demonstrates a physiological function for DIAP1, the control of SOP cell development, in a nonapoptotic role through the control of caspase activation. Roles of caspases in nonapoptotic situations have been studied in mammals (reviewed in Schwerk, 2003). Recently, some groups have reported that caspases and caspase regulators, likely acting at distinct points in time and space, are required for nonapoptotic processes in Drosophila. Arama (2003) and Huh (2004) demonstrated that caspases are required for spermatid individualization, the process in which haploid syncytial spermatids are differentiated into individual motile sperm. Inhibition of the GTPase Rac induces a defect in border-cell migration in the Drosophila ovary, and this defect is suppressed by the coexpression of DIAP1 or the reduction of caspase activity (Geisbrecht, 2004). Furthermore, the nonapoptotic caspase activation must clearly be tightly regulated to prevent cells from dying inappropriately through apoptosis. It seems likely that DmIKKε controls the quantity of DIAP1 in some cells, such as proneural clusters, thereby determining the level of caspase activity, which is required for some nonapoptotic processes. DmIKKε also regulates the actin cytoskeleton through the DIAP1/caspase pathway (Oshima, 2006). Interestingly, it has also been demonstrated that the mammalian IKK-related kinase NAK can phosphorylate XIAP. The IKK-related kinase can be activated in mammals in response to not only immune stimuli but also growth factors such as PDGF (Tojima, 2000), suggesting that IKK-related-kinase-mediated IAP regulatory functions might be involved in the immune system, as well as in development, through caspase activity that is required for nonapoptotic processes (Kuranaga, 2006).

DmIKKε accelerates DIAP1 degradation in a kinase-dependent manner. This suggests that the DmIKKε-induced phosphorylation of DIAP1 is required to turn on the destruction switch and activate physiological phenomena that are normally inhibited by DIAP1. Different mechanisms of DIAP1 regulation have been proposed, including the promotion of the autoubiquitination of DIAP1 by RHG (Rpr, Hid, and Grim) proteins, the N-end rule (that relates the in vivo half-life of a protein to the identity of its N-terminal residue), and transcription. The RHG proteins are not required for DmIKKε-induced DIAP1 degradation. Regarding the N-end rule, a CHX chase experiment was performed using DRONC dsRNA in S2 cells and it was confirmed that the N-end rule pathway positively regulates endogenous DIAP1 turnover in S2 cells. Hippo (Hpo) is a serine/threonine kinase belonging to the Ste20-like family of kinases that has been implicated in DIAP1 inhibition. Two different mechanisms of DIAP1 inhibition by Hpo have been proposed, one transcriptional and the other posttranslational. Therefore whether the Hpo-induced phosphorylation of DIAP1 is involved in the degradation of DIAP1 that is induced by Rpr or DmIKKε was tested. When DIAP1 was mutated at the site where Hpo is predicted to phosphorylate (Ser159, Ser164, Thr114, or Thr115), it did not provide resistance against Rpr- or DmIKKε-induced degradation, suggesting that the direct phosphorylation of DIAP1 by Hpo is not involved in DIAP1 degradation under the experimental conditions used. A recent report provides evidence that Hpo regulates the transcription of DIAP1 rather than directly phosphorylating it (Huang, 2005). The knockdown of DmIKKε in S2 cells causes the accumulation of endogenous DIAP1 but does not affect the diap1 mRNA level. Taken together, these findings indicate that DmIKKε is a bona fide kinase that can regulate DIAP1 degradation via phosphorylation (Kuranaga, 2006).

A transport and retention mechanism for the sustained distal localization of Spn-F-IKKε during Drosophila bristle elongation

Stable localization of the signaling complex is essential for the robust morphogenesis of polarized cells. Cell elongation involves molecular signaling centers that coordinately regulate intracellular transport and cytoskeletal structures. In Drosophila bristle elongation, the protein kinase IKKε is activated at the distal tip of the growing bristle and regulates the shuttling movement of recycling endosomes and cytoskeletal organization. However, how the distal tip localization of IKKε is established and maintained during bristle elongation is unknown. This study demonstrates that IKKε distal tip localization is regulated by Spindle-F (Spn-F), which is stably retained at the distal tip and functions as an adaptor linking IKKε to cytoplasmic dynein. Javelin-like (Jvl) is a key regulator of Spn-F retention. In jvl mutant bristles, IKKε and Spn-F initially localize to the distal tip but fail to be retained there. In S2 cells, particles that stain positively for Jvl or Spn-F move in a microtubule-dependent manner, whereas Jvl and Spn-F double-positive particles are immobile, indicating that Jvl and Spn-F are transported separately and, upon forming a complex, immobilize each other. These results suggest that polarized transport and selective retention regulate the distal tip localization of the Spn-F-IKKε complex during bristle cell elongation (Otani, 2015).

Highly polarized cells, such as neurons and epithelial cells, rely heavily on intracellular transport mechanisms for their functional differentiation. Disrupted intracellular transport systems lead to a variety of diseases, including neurodegeneration and microvillus inclusion diseases. Accurate intracellular transport is ensured by the polarized cytoskeleton and by the adaptor protein-mediated recognition of specific cargoes by molecular motors. Molecular motors play central roles in intracellular transport, and have diversified through evolution. However, the diversity of molecular motors is not sufficient to explain transport specificity, as various cargoes often share the same motor while being transported to distinct locations. For example, cytoplasmic dynein is the major microtubule minus-end motor and transports a variety of cargoes including the Golgi apparatus, endosomes and RNAs. Evidence suggests that the fate of cargo is determined not only by cargo-motor recognition, which occurs upon cargo loading, but also at the cargo destination site. For instance, in axonal transport some cargoes, such as dense core vesicles and synaptic vesicles, are inefficiently captured at synaptic boutons and circulate within the axon, whereas others, such as mitochondria, are stably retained at synapses. Although the precise regulation of cargo transport is important for the functional differentiation of various polarized cells, the underlying molecular mechanisms remain poorly understood (Otani, 2015).

Cell elongation is a widely observed morphogenetic event that requires the coordinated input of intracellular transport, the cytoskeleton and cell polarity. Drosophila bristles, which are hair-like unicellular structures that function as external sensory organs, are formed by the elongation of trichogen cells, which can grow up to 350 μm in 1 day during the pupal stage. IκB kinase ε [IKKε; also known as IκB kinase-like 2 (Ik2)] acts at the distal tip of growing bristles and functions as a signaling center to regulate the bidirectional shuttling of Rab11-positive recycling endosomes during bristle elongation. Rab11-positive vesicles are transported to the distal tip by interacting with cytoplasmic dynein via an adaptor protein Nuf/Rab11FIP3. At the distal tip, IKKε phosphorylates Nuf to inactivate dynein-dependent trafficking, thereby promoting the directional switching of the recycling endosomes. In addition to its role in endosome trafficking, IKKε regulates the organization of both actin and microtubules. However, how IKKε is localized to the distal tip of growing bristles is unknown (Otani, 2015).

Spindle-F (Spn-F) is a coiled-coil protein that interacts with IKKε and has been implicated in regulating IKKε polarized activation. In oocytes, the intracellular localizations of Spn-F and IKKε depend on each other, and spn-F and ikkε mutants show similar bristle morphology and oocyte polarization phenotypes, suggesting that they function together. Several proteins other than IKKε, including Cut up (Ctp)/dynein light chain (LC8) and Javelin-like (Jvl), are reported to interact with Spn-F. It was proposed that Spn-F interacts with cytoplasmic dynein via Ctp to localize the Spn-F-IKKε complex to microtubule minus ends. However, subsequent structural studies indicated that Ctp/LC8 cannot simultaneously bind dynein and cargo molecules, challenging this model. On the other hand, IKKε can phosphorylate Spn-F, suggesting that Spn-F might act downstream of IKKε. Interestingly, another Spn-F-interacting protein, Jvl, was recently shown to regulate the polarized activation of IKKε in oocytes. Although Jvl can interact with microtubules, how it regulates the polarized activation of IKKε is unknown (Otani, 2015).

This study demonstrated that the bristle tip is a sorting station for cytoplasmic dynein-dependent cargoes. The IKKε-Spn-F complex, which acts as the signaling center in bristle cell elongation, localizes to the distal tip by dynein-dependent polarized transport and Jvl-dependent selective retention. By contrast, Rab11-positive recycling endosomes undergo both dynein-dependent distal transport and proximal transport, which is probably mediated by kinesins (Otani, 2015 and references therein).

The distinct transport characteristics at the distal tip are specified by the nature of the adaptor proteins. IKKε is transported to the distal tip by dynein via the adaptor protein Spn-F, and the IKKε-Spn-F complex is stably retained at the distal tip by Jvl, a Spn-F-interacting protein. By contrast, Rab11-positive recycling endosomes are transported to the distal tip by dynein via the adaptor protein Nuf, where it is phosphorylated by IKKε. This phosphorylation inactivates the dynein-dependent transport of Rab11-positive recycling endosomes, thereby promoting their transport back to the cell body. Thus, the IKKε-Spn-F complex stably localizes to the distal tip by polarized transport followed by selective retention, whereas Rab11-positive recycling endosomes bidirectionally shuttle by polarized transport and motor switching. The pivotal step in this sorting decision is the specific recognition of the cargo adaptor proteins (Spn-F and Nuf) by their regulatory proteins (Jvl and IKKε) at the distal tip. These results support the emerging concept that cargo adaptor proteins are not merely physical linkers between cargoes and motors, but act as regulatory hubs where various signals converge (Otani, 2015).

This study identified Jvl as a key regulator of IKKε-Spn-F retention at the distal tip. Jvl interacts with microtubules, and binding Spn-F promotes the microtubule binding activity of Jvl and induces microtubule bundling in S2 cells. Full-length Jvl localizes to punctate structures that were located along microtubules, whereas the C-terminal half of Jvl uniformly decorated microtubules. These results imply that Jvl microtubule binding activity is repressed by its N-terminal region, and that binding Spn-F could relieve this inhibition. Oligomerization of Spn-F could promote the formation of higher-order Spn-F-Jvl complexes to generate multivalent microtubule-binding sites, thereby increasing the microtubule binding activity of Jvl (Otani, 2015).

Spn-F and Jvl are independently transported to the distal tip in elongating bristles, indicating that their interaction occurs upon arrival at the tip. This interaction presumably activates Jvl microtubule binding activity, which then serves as a molecular brake to immobilize the complex on microtubules. Similar mechanisms have been proposed for the anchoring of mitochondria by Syntaphilin and Kinesin-1 in axonal mitochondrial transport, and for the immobilization of lysosomes in dendrites by the interaction of TMEM106B and MAP6. The coupling of cargo adaptor proteins with microtubule-binding proteins might be a general mechanism for regulating the transport of a particular cargo in a spatiotemporally controlled manner. As Spn-F and Jvl are also involved in the polarized activation of IKKε during oogenesis, similar mechanisms might help generate and maintain cell polarity in various cell types (Otani, 2015).

As an alternative to the molecular brake model, Jvl could act as a scaffolding protein to recruit enzymes that modify the IKKε-Spn-F complex to promote its retention, or as a regulator of microtubule organization at the distal tip to maintain the polarized organization of the cytoskeleton during bristle elongation. Further analysis of the molecular functions of Jvl will help in elucidating the mechanisms of IKKε-Spn-F retention (Otani, 2015).

The results suggest that Spn-F functions as a cargo adaptor for IKKε and cytoplasmic dynein. Structure-function analysis of Spn-F demonstrated that its dynein-binding region is required for localizing IKKε to the distal tip and for bristle morphogenesis. In contrast to the dynein-binding-deficient Spn-F mutant, which partially suppressed the spn-F mutant bristle morphology phenotype, a mutant lacking the IKKε-binding region completely failed to rescue, indicating that, in addition to its function as a cargo adaptor, Spn-F has a role in regulating IKKε activity. This role could involve regulating IKKε kinase activity or protein stability, or in scaffolding the components of the IKKε signaling pathway. IKKε overexpression could partially suppress the spn-F mutant bristle morphology phenotype despite IKKε delocalization from the distal tip, suggesting that increasing the dosage of IKKε can compensate for the loss of Spn-F to some extent. It is likely that the delocalized IKKε can phosphorylate some of its downstream target molecules (such as Nuf and Diap1) to partially support bristle morphogenesis (Otani, 2015).

In summary, this study has demonstrated that the signaling center for bristle elongation is localized to the distal tip by polarized transport and selective retention mechanisms. The distal tip of bristles acts as a sorting center for cytoplasmic dynein cargoes, where regulatory proteins recognize cargo adaptor proteins and determine whether cargo is retained or sent back to the cell body. These findings support the idea that cargo adaptor proteins act as regulatory hubs where various signals converge. It would be interesting to test whether the differential regulation of cargo-motor interactions contributes to the formation of signaling centers during the morphogenesis of mammalian cells of complex shape, such as neurons and podocytes (Otani, 2015).

REGULATION

Protein Interactions

Whether physical binding occurs between endogenous DmIKKε and DIAP1 was tested. An immunoprecipitation experiment was performed against the endogenous DmIKKε protein using an anti-DmIKKε monoclonal antibody. The specificity of this antibody was confirmed by immunoblotting to detect endogenous DmIKKε protein in normal and DmIKKε knockdown cells. These results indicated that physical binding occurs between endogenous DmIKKε and endogenous DIAP1 in S2 cells. When the S2 cells were treated with DmIKKε dsRNA, endogenous DIAP1 protein was not immunoprecipitated by the DmIKKε antibody. To elucidate the function of the ubiquitin-like (Ubl) domain of DmIKKε, the binding of a deletion mutant for this domain was tested. DmIKKεΔUbl interacted weakly with DIAP1, whereas wild-type or kinase-dead mutants of DmIKKε significantly bound to DIAP1. Consistent with the binding assay, DmIKKεΔUbl did not cause cell death or DIAP1 degradation in S2 cells. The binding of purified DIAP1 and DmIKKε was not detected by a GST pull-down assay in vitro, indicating that this physical binding may not be direct. It is likely that DmIKKε phosphorylates DIAP1 in a protein complex in cells and that the Ubl domain of DmIKKε is required for efficient formation of the DmIKKε/DIAP1 complex (Kuranaga, 2006).

Whether DIAP1 degradation might be regulated by DmIKKε-induced phosphorylation was tested. Wild-type DmIKKε was autophosphorylated and induced the phosphorylation of DIAP1 in S2 cells, whereas none of the four mutant DmIKKεs generated from the alleles DmIKKε^G19R, DmIKKε^D160N, DmIKKε^G250D, or DmIKKεΔUbl did so. Moreover, the kinase-dead DmIKKε mutants did not reduce DIAP1 levels or cause cell death in S2 cells, and they did not cause eye ablation in adult flies (Kuranaga, 2006).

Whether the human IKK-related kinase NAK could also elicit DIAP1 degradation was tested. The expression of human NAK in S2 cells strongly induced cell death as well as DIAP1 degradation, whereas a kinase-dead form of NAK, NAK^K41M, did not. These data imply that DIAP1 degradation mechanisms are conserved between mammalian IKK-related kinase and DmIKKε. To investigate whether mammalian IAP could be phosphorylated by mammalian IKK-related kinase, XIAP phosphorylation and degradation by NAK were tested. When 293T cells were cultured in media with 10% serum, a band shift was observed but not reduction of XIAP protein. XIAP band shift was cancelled by phosphatase treatment. The same experiments were performed under low-serum conditions (1%), and it was found that NAK expression induced both a band shift of XIAP and the reduction of XIAP protein. it was also observed that NAK phosphorylated XIAP in vitro, suggesting that the function of IKK-related kinase in the phosphorylation and degradation of IAP is conserved in both Drosophila and mammalian cells (Kuranaga, 2006).

spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila

The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfα-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. The ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. spn-F was clond and found to encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. Spn-F interacts directly with the Dynein light chain Ddlc-1 (Cut up). These results show that this novel protein affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis (Abdu, 2006; full text of article).

In a global two-hybrid screen, Spn-F (CG12114) was found to interact with the Ik2 (CG2615) protein. Mutations in Ik2 have been isolated and characterized, ik2 mutants share many phenotypes with spn-F, including a very similar bristle phenotype and specific effects on MT organization in oogenesis. However, ik2 mutants are lethal, whereas spn-F homozygotes survive. In addition, whereas spnF mutations have only mild effects on Oskar protein localization and a low frequency of bicaudal phenotypes, such effects are more pronounced in the ik2 mutants. Nevertheless, the striking similarities strongly suggest that the two genes function in a common pathway that affects certain types of MT more strongly than others. In normal mitotic cells, the minus ends of MTs are usually focused by the centrosomes in the interior of the cell, and plus ends contact the cortex. However, in specialized cells, such as the Drosophila oocyte, there are minus ends that make contact with the cortex. It is therefore possible that Spn-F and Ik2 are required for providing a stable connection between such cortical MT minus ends and cortical actin for subsets of MTs involved in specialized transport processes. Future experiments will address the interactions of Spn-F and Ik2 directly, and will determine whether, for instance, Spn-F might be a target of Ik2 (Abdu, 2006).

The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant (Abdu, 2006), whether Spn-F could be a direct regulatory target of Ik2 was investigated. It was found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. Ik2 phosphorylates Spn-F and this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization. It is concluded that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation (Dubin-Bar, 2008).

Recent studies implicate the Drosophila IKKε homologue ik2 in seemingly unrelated NF-κB functions. It has been shown that ik2 modulates caspases for a nonapoptotic function and controls both actin and MT cytoskeletons, and also that it regulates the actin cytoskeleton through phosphorylation and degradation of DIAP1. Moreover, in ik2 mutant oocytes, abnormal mRNA localization can be attributed to defects in organization of MT minus-ends, giving rise to ventralized and bicaudal phenotypes of mutant embryos. Whereas the control of actin polymerization appears to be mediated by a nonapoptotic function of DIAP1, the regulatory target of Ik2 in controlling cytoskeleton organization in the oocyte is still unknown. This study examined whether spn-F, which showed precisely the same ovarian and bristle phenotypes as ik2 mutants, might be an Ik2 target. In previous work has shown that spn-F encodes a novel protein that affects oocyte axis determination and the organization of MTs during Drosophila oogenesis. This work shows that Ik2 physically interacts with Spn-F and forms a relatively stable complex. In addition, Ik2 phosphorylates Spn-F, but the interaction between these two proteins is independent of phosphorylation. Thus the results suggest that Spn-F is a putative regulatory substrate of Ik2. Moreover, the results indicate that the nature of the interaction between Spn-F and Ik2 is different from that attributed to Ik2 and DIAP1. Spn-F phosphorylation by Ik2 has no effect on its stability in S2 cells. Supporting this conclusion is the finding that over-expression of Ik2 in the ovaries has no effect on Spn-F stability or development, indicating that over-expression of ik2 does not lead to Spn-F degradation. Furthermore, the ovarian phenotype of spn-F mutant is similar to the ovarian phenotype of ik2. Thus, whereas Ik2 regulates organization of the actin cytoskeleton via phosphorylation and degradation of DIAP1, in the oocyte, rather than affecting Spn-F degradation, Ik2 and Spn-F form a complex that regulates the oocyte cytoskeleton (Dubin-Bar, 2008).

How does the IK2/Spn-F complex function in the germline? This study showed that Ik2 and Spn-F are co-localized both to the anterior ring during mid-oogenesis and to punctate structures in the nurse cells. Additionally, Ik2 and Spn-F are mutually required for correct localization in the germline. In ik2 germline clones, Spn-F protein localization along the anterior cortex is significantly reduced relative to the wild-type egg chamber and Spn-F aggregates in the oocyte; also, there is a higher accumulation of the punctate structures containing Spn-F protein in the nurse cells as compared with the wild type. Furthermore, Ik2 localization to the anterior end of the oocyte and to the punctate structure in the nurse cells depends on spn-F. Thus, it is suggested that the correct localization of Spn-F and Ik2 complex to special compartment within the oocyte is an essential requirement for organization of oocyte cytoskeleton. The defects observed in ik2 and spn-F mutants oocyte are most likely due to misslocalization of the Ik2/Spn-F complex (Dubin-Bar, 2008).

Immunostaining has shown that Spn-F protein localizes to the minus end of the MT network in the oocyte and also to granules in the nurse cells. Previous work has shown that depolymerization of MT and mutations in Dynein heavy chain cause a significant loss of Spn-F localization at the oocyte anterior cortex. In addition, these treatments results in substantial increase in both the number and the size of Spn-F granules in the nurse cells. These observations suggest that Spn-F is transported from the nurse cells to the oocyte and that this transport could be mediated by Dynein. The present study found that when Ik2 was expressed alone in S2R+ cells, it was evenly distributed in the cytoplasm whereas Spn-F localized to cytoplasmic punctate structures. When both proteins were co-expressed in S2R+, Ik2 was co-localized to the cytoplasmic punctate structures along with Spn-F. Moreover, a higher accumulation of punctate structures containing Spn-F protein was found in the nurse cells in ik2 mutants as compared with the wild type, similarly to what is observed in the Dynein heavy chain mutant. Taking all of these results into account, it is proposed that Spn-F is required for localization of Ik2 to cytoplasmic transport vesicles while Ik2 is required for correct transport of the complex from nurse cells to oocyte. Once the complexes are in the oocyte, they may accumulate at certain cortical sites where they promote the interaction of MTs and the actin cytoskeleton (Dubin-Bar, 2008).

In conclusion, Ik2 and Spn-F form a complex which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Unlike other IKK proteins, Ik2 phosphorylates Spn-F without promoting its degradation (Dubin-Bar, 2008).

EFFECTS OF MUTATION

IKK epsilon regulates F actin assembly and interacts with Drosophila IAP1 in cellular morphogenesis

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKKε is a member of the IκB kinase family, and its developmental role has not been clear. Drosophila IKKε localizes to the ruffling membrane of cultured cells and is required for F actin turnover at the cell margin. In IKKε mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibit aberrantly branched morphology. These phenotypes are sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKKε destabilizes F actin-based structures. Expression of a dominant-negative form of IKKε increases the amount of DIAP1. The results suggest that at the physiological level, IKKε acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKKε function involves the negative regulation of the nonapoptotic activity of DIAP1 (Oshima, 2006).

In a gene misexpression screen for regulators of epithelial morphogenesis of the Drosophila tracheal system, IKKε (CG2615), was identified a member of the IKK protein kinases that are known to activate NFκB. IKKε and TBK1 form a distinct subfamily of IKKs (Peters, 2000: Kishore, 2000), and their roles in development remain unclear. The tracheae consist of tubular epithelium whose apical surface labeled with the F actin marker GFP-moesin faces the luminal side. Overexpression of IKKε disrupts epithelial integrity, and the prominent apical accumulation of F actin and apical-basal polarity are both lost with no apparent sign of cell death. IKKε mRNA is detected ubiquitously, and the IKKε protein is present as punctate patterns in the tracheal terminal branch (Oshima, 2006).

To reveal the cellular functions of IKKε, its role was studied in Drosophila S2 cells. IKKε is concentrated in the thick cytoplasmic (C) domain. In the thin peripheral (P) domain, a retrograde F actin flow is active and occasionally forms a ruffling membrane where IKKε and F actin colocalize, suggesting that the localization of IKKε and F actin is, in general, mutually exclusive, but that they colocalize where F actin-driven cell motility is high. IKKε activity is reduced by transfecting a dominant-negative IKKε (IKKε^DN: a kinase-defective K41A mutation) or by double-stranded-RNA-mediated knockdown. Neither of these treatments altered cell viability. The cell shapes of S2 cells were grouped into three classes—stellate, serrate, and smooth—and the frequency of each class was quantified (morphology index: MI). The MI of IKKε^DN or IKKε^RNAi cells was higher than that of control cells, suggesting a possible involvement of IKKε in F actin-based cell morphogenesis. The function of IKKε in F actin dynamics was studied. The kymograph showed the distinct P domain with parallel diagonal lines, indicating a constant retrograde flow of F actin, and the C domain with horizontal lines, indicating a static F actin. In serrate-class cells with IKKε^DN, a slight deceleration of the retrograde F actin flow was observed , as well as a narrowing of the P domain. P domain size decreased from 6.70 ± 2.38 μm in control cells to 4.37 ± 1.67 μm in IKKε^DN-expressing cells, suggesting that IKKε is required for active retrograde F actin movement and to maintain the flat cell shape (Oshima, 2006).

To determine the effect of an elevated level of IKKε on cell shape, cells were stably transformed with a methallothionein-promoter-driven IKKε construct. By controlling the level of induction, it was possiable to establish a condition that did not alter cell viability assessed by measuring transfected lacZ activity. It was found that the MI of IKKε-expressing cells was lower than that of the control cell line and that the rate of retrograde F actin flow was increased, suggesting that a moderate elevation of the level of IKKε affects cell shape and F actin dynamics. It was also noted that a high level of IKKε activity caused stochastic entry into apoptosis. By selecting viable cells by replating onto Con A-treated plates, it was found that MI was reduced and the retrograde F actin flow and membrane ruffling were increased. These results suggest that IKKε can both alter cell shape and promote F actin flow. It was also found that IKKε affects the dynamics of microtubule plus ends in the P domain. Because this effect was cytochalasin D sensitive, it was concluded that IKKε indirectly regulates microtubule plus-end dynamics in the P domain by promoting retrograde F actin flow (Oshima, 2006).

The phenotype of the embryonic tracheal system was examined in the strong loss-of-function mutant ikkε⁶⁶. Tracheal branching occurred normally with apparently normal cell number. In stage 17 embryos, however, the terminal branches displayed abnormal morphologies. One class was the duplication of terminal cells expressing the terminal-cell marker SRF. Because of the level of activated MAP kinase being indistinguishable from that of the control, the level of FGF signaling appeared to be unaltered. The other classes showed the phenotype of turned or bifurcated actin cores in terminal cells. Focus was placed on these classes to address the role of IKKε in cell shape (Oshima, 2006).

The terminal branch consists of a single terminal cell that contains distinct F actin-rich structures, the central actin core, F actin dots, and peripheral filopodia. Control terminal cells initially take on a bipolar shape, extending numerous filopodia in both dorsal and ventral sides, with the actin core often having a U shaped structure, bending toward the dorsal margin, where F actin dots and filopodia are abundant. During the next 30 min period, terminal cells undergo a change from a bipolar to monopolar shape, filopodia in the dorsal half decrease, and the actin core is redirected ventrally. Throughout this process, F actin dots transiently align near the growing distal tip of the actin core and later mature into the actin core (Oshima, 2006).

In the 'turned' class of ikkε⁶⁶ mutant cells, the distal tip of the actin core remains directed toward the dorsal side. Although no significant difference was detected in the stability of individual filopodia, filopodia extension persists in both the dorsal and ventral side in ikkε⁶⁶ mutants. The number of F actin dots increases in the ikkε⁶⁶ mutants, and a similar phenotype is observed by the tracheal-specific expression of IKKε^DN. Furthermore, ikkε^RNAi causes the terminal branch to turn or duplicate, suggesting cell autonomy of IKKε function. Additionally, palm-like structures with secondary and tertiary branches at the tips of filopodia are frequently observed in ikkε⁶⁶ mutants. It is concluded that the loss of IKKε function leads to persistent filopodia formation in the dorsal side and the ectopic accumulation of F actin dots, as well as to the misorientation and bifurcation of the actin core and the terminal branch (Oshima, 2006).

Sensory bristles are formed by the outgrowth of shaft cells into the animal's exterior, accompanied by the assembly of a small cluster of F actin filaments close to the membrane of the growing tip. IKKε function was inhibited with IKKε^DN or dsRNA interference (ikkε^RNAi), the macrochaetae became gnarled and forked. Inhibition of IKKε also causes malformation of the antenna arista, a terminal segment of the antenna composed of several unicellular laterals and a multicellular central core. This phenotype resembles that of actin-regulatory gene mutants, suggesting that IKKε prevents excess branching through regulation of F actin asembly (Oshima, 2006).

Another class of genes involved in arista morphogenesis includes the cell-death regulators hid and Drosophila inhibitor of apoptosis protein 1 (DIAP1). The identification of DIAP1 as a negative regulator of IKKε (Kuranaga, 2006) led to an investigation of the genetic interaction of DIAP1 and IKKε. The dosage of DIAP1 was modified by dsRNAi (DIAP1^RNAi) or by overexpression (DIAP1^OE), which did not cause a significant change in apoptosis and normal number and morphology of arista was allowed. The excess-branching phenotype of IKKε^DN was suppressed by DIAP1^RNAi and enhanced by DIAP1^OE. IKKε^DN also enhances the overexpression phenotype of Profilin, suggesting that IKKε is involved in regulation of F actin in laterals. In S2 cells, DIAP1^OE increases the frequency of the serrate/stellate class of cells. The coexpression of DIAP1 and IKKε^DN has an additive effect, and coexpression of DIAP1 with IKKε recovers cell viability from ~20% to ~50% (assessed by transfected lacZ activity). Remarkably, the cell morphological effect of IKKε overexpression is completely suppressed by DIAP1^OE. Mild reduction of the regulatory caspase DRONC and its activator DARK by RNAi enhanced the lateral phenotype of IKKε^DN. In S2 cells, reduction of DRONC by RNAi or by expression of the specific inhibitor p49 increased MI. Inhibition of effector caspases by p35 had no effect on morphologies of laterals and S2 cells, suggesting that a simple obstruction of effector caspases is insufficient in changing cell shape. In addition, overexpression of IKKε inhibits migration of border cells in the ovary without apparent evidence of apoptosis, a phenotype similar to DIAP1 mutants. Finally, IKKε^DN increased the level of DIAP1 by 3.3-fold (Oshima, 2006).

These results indicate that IKKε and DIAP1 have opposing effects on the shape of arista laterals and S2 cells, and that DIAP1 acts in parallel with, or downstream of, IKKε. It is suggested that IKKε prevents the inappropriate accumulation of F actin and thereby functions as a proofreading mechanism in morphogenesis when high precision is required for the monopolar growth of cell protrusions. IKKε also functions in the polarization of Drosophila oocytes (Shapiro, 2006; Oshima, 2006).

Kuranaga (2006) recently demonstrated that IKKε promotes degradation of DIAP1. IKKε is ubiquitously expressed in cells without any sign of apoptosis, and reduction of IKKε did not apparently affect cell viability, suggesting that the normal level of IKKε is insufficient to promote apoptosis and that the major physiological role of IKKε is to modulate cell shape. Given that the cell-shape activity of IKKε interacts with DIAP1 and DRONC, these apoptosis regulators may also play nonapoptotic roles in this context by physically interacting with Profilin or by cleaving critical regulators of F actin dynamics. The identification of IKKε provides a strong starting step toward clarifying the nonapoptotic functions of DIAP1 and Caspases in cellular morphogenesis (Oshima, 2006).

DmIKKε Promotes DIAP1 elimination and cell death

Caspase-dependent cell death is regulated by the expression of proapoptotic proteins such as Reaper (Rpr) that promote DIAP1 degradation, leading to caspase-dependent cell death. Thus, it should be possible to gain insight into the physiological functions of DIAP1 by identifying regulators of DIAP1 inhibition using a Rpr-induced phenotype (GMR-RprS). To identify the regulators of DIAP1, a dominant-modifier screen was performed for Rpr-induced eye ablation using a set of deficiency strains that cover more than 70% of the Drosophila genome, as described previously. Fifteen mutant lines were identified and named apoptoxin (APTX) for their putative proapoptotic function. Df-APTX7, which has deletions uncovering 38A6-40A1, were identified as a suppressor of Rpr. In the secondary screen using small deficiencies, the overlapping suppressor region as 38C3-38F2 were identified. Next, flies were tested with known mutations in the 38C3-38F2 region for their ability to improve the Rpr-induced small-eye phenotype. Three lines corresponding to APTX7 (APTX7³⁶, APTX7⁴⁶, and APTX7⁶⁶) improved the Rpr-induced phenotype. Interestingly, the CG2615 gene in the same region was also identified as a candidate gene in a misexpression screen for cell-death executioners (endd 29; Kanuka, 2005a); therefore, the cDNA sequence of CG2615 was determined in each of the three APTX7 alleles (Kuranaga, 2006).

Because DmIKKε is a candidate component in the pathway of Rpr-induced cell death, it is possible that DmIKKε might act as a proapoptotic protein. To investigate this possibility, Drosophila S2 cells were transfected with expression vectors for DmIKKε. DmIKKε strongly induced cell death, like Rpr. Moreover, the generation of DmIKKε-expressing transgenic flies showed that DmIKKε is a cell-death inducer because its ectopic expression in the fly compound eye shows a severe eye-ablation phenotype, which is similar to the Rpr phenotype. To verify that the DmIKKε-induced small-eye phenotype is caused by cell death due to caspase activation, the TUNEL assay and fluorescence resonance energy transfer (FRET) analysis were used to detect caspase activity in the eye discs. The overexpression of DmIKKε driven by GMR-GAL4 increased the number of TUNEL-positive cells in the posterior region of the morphogenetic furrow of eye discs. To determine the caspase activity in vivo, SCAT3, a FRET indicator for caspase activity was used. In the eye discs of SCAT3-expressing flies with the GMR-GAL4 driver, the coexpression of DmIKKε caused caspase activation (FRET abolishment). The cell shape in the eye discs of DmIKKε-expressing flies was carefully observed and it was noticed that many cells formed apoptotic bodies in these discs. It was further verified that the DmIKKε-induced small-eye phenotype was caused by cell death due to caspase activation because coexpression of a dominant-negative form of DRONC (DRONC DN) partially improved the DmIKKε-induced eye-ablation phenotype. These data support the idea that DmIKKε's ectopic expression induced cell death via caspase activation in the Drosophila eye. The observation that the coexpression of a caspase inhibitor could not completely rescue the rough-eye phenotype of DmIKKε-expressing flies, unlike its effect in Rpr-expressing flies, suggested that overexpressed DmIKKε might cause developmental defects or stress in the eye discs in addition to cell death (Kuranaga, 2006).

The possibility was considered that DmIKKε affects the degradation of DIAP1 because the strength of the cell-death induction by DmIKKε was similar to that of Rpr, which induces cell death by promoting DIAP1 degradation. In fact, the endogenous DIAP1 protein level is significantly decreased in the DmIKKε-expressing region, as indicated by anti-GFP staining. Since DIAP1 degradation is promoted by the RHG (Rpr, Hid, and Grim) proteins, it is possible that DmIKKε induced the transcriptional upregulation of RHG mRNAs, resulting in DIAP1 degradation and cell death. An H99 clone that has a deletion uncovering the RHG genomic region was used to investigate the genetic interaction between DmIKKε and the RHG. DmIKKε-induced cell death and DIAP1 reduction were not suppressed in H99 mutant clones of eye discs. Consistent with this result, the expression of DmIKKε did not induce the upregulation of RHG mRNA in larval eye discs. These results indicated that DmIKKε induce DIAP1 reduction and cell death independently of the RHG proteins. To verify that DmIKKε regulates DIAP1 degradation, S2 cells were cotransfected with DIAP1 and DmIKKε. DmIKKε expression induced the degradation of exogenous FLAG-tagged DIAP1 protein in a dose-dependent manner. DmIKKε did not affect the expression level of FLAG-diap1 mRNA. These results suggest that the DmIKKε-induced DIAP1 reduction is mediated by the posttranslational degradation of DIAP1. Whether the DmIKKε-induced DIAP1 degradation is inhibited by treatment with the proteasome inhibitor lactacystin was tested, and it was confirmed that the DmIKKε-induced reduction in DIAP1 is prevented by the proteasome inhibitor in S2 cells. This finding suggests that the DmIKKε-induced DIAP1 degradation is mediated by the proteasome pathway (Kuranaga, 2006).

DmIKKε acts as a regulator for endogenous DIAP1 turnover

It is likely that endogenous DmIKKε functions to regulate endogenous DIAP1 protein turnover. The endogenous DIAP1 level increases when the endogenous DmIKKε level is decreased in S2 cells by treatment with DmIKKε dsRNA. Knockdown of DmIKKε in S2 cells did not affect the diap1 mRNA level, as examined by semiquantitative RT-PCR. To examine whether DIAP1 turnover is regulated by DmIKKε in S2 cells, cycloheximide (CHX) chase experiments were performed. It was confirmed that the endogenous DIAP1 had a half-life of about 30 min in S2 cells. However, when DmIKKε expression was reduced in S2 cells, DIAP1 protein turnover was significantly delayed after CHX treatment. These data strongly support the idea that DmIKKε regulates endogenous DIAP1 protein turnover (Kuranaga, 2006).

DmIKKε Regulates caspase activation through regulation of DIAP1 level without affecting developmental cell death

DmIKKε's regulation of the endogenous DIAP1 level was examined in vivo. Since homozygous DmIKKε mutants are usually embryonically lethal and the escapers die before the second larval stage, and since MARCM (mosaic analysis with a repressible cell marker) was unsuccessful due to a very low efficiency of recombination, RNA interference (RNAi) was used to analyze the DmIKKε loss-of-function phenotype in vivo. Transgenic animals were generated bearing an inverted repeat (IR) fragment of the DmIKKε cDNA in the pUAST vector (DmIKKε IR). Overexpression of the DmIKKε IR induced a reduction in the coexpressed DmIKKε protein level in S2 cells and suppressed the DmIKKε-induced phenotype in fly eyes. Then DmIKKε knockdown lines were used for a mosaic experiment. The subset of DmIKKε knockdown cells, which were labeled with GFP, exhibited stronger DIAP1 signal in the wing discs than did cells with normal DmIKKε levels, indicating that DmIKKε determines the endogenous level of DIAP1 protein (Kuranaga, 2006).

Considering that the function of DIAP1 is to inhibit caspase activation, it is possible that the physiological role of DmIKKε is to control caspase activity and cell death via regulation of the DIAP1 protein level. No significant changes were seen in embryonic cell death in homozygous or transheterozygous DmIKKε mutants. However, since the maternal DmIKKε contribution might affect the cell death in DmIKKε mutants during the early embryonic stage, TUNEL staining was performed in the DmIKKε knockdown wing discs. A large amount of naturally occurring cell death is observed in the wing disc of third-instar larvae, and this cell death was not suppressed in the clone or in the DmIKKε knocked-down posterior region. It is possible that, while cell-death stimuli such as Rpr, Hid, and Grim promote cell death through strong DIAP1 degradation, DmIKKε-mediated determination of the DIAP1 level through the regulation of DIAP1 turnover might not be a major mechanism regulating developmental cell death (Kuranaga, 2006).

Caspase activity without cell death controls various physiological processes, including cell differentiation; however, the regulatory mechanisms of caspase activity in this context are largely unknown. With this in mind, the effects of DmIKKε knockdown on caspase activity was examined in the absence of cell-death stimuli. Generally, an anti-active caspase antibody is used to stain dying cells. However, it is very hard to detect low or mild levels of caspase activity in cells by this method. Attempts were made to detect caspase activation using an anti-active caspase antibody in the wing discs, but its positive signals were few and scattered. Therefore, to determine the caspase activity in vivo, the caspase indicator SCAT3 was used. Interestingly, the FRET abolishment was weak in a broad area of the wild-type wing discs, suggesting that caspase was mildly activated in wing discs even in nonapoptotic cells. This mild activation of caspase was suppressed by DmIKKε knockdown. Endogenous DEVDase activity was also detected in S2 cells and was suppressed when endogenous DIAP1 accumulated following treatment with DmIKKε dsRNA. These results imply that DmIKKε controls endogenous caspase activity via quantitative regulation of the DIAP1 protein and that this mechanism might be involved in the regulation of nonapoptotic caspase functions (Kuranaga, 2006).

DmIKKε Is involved in macrochaete formation through control of caspase activation

The number of macrochaetae forming in the scutellum of the notum is determined through a nonapoptotic function of caspases (Kanuka, 2005b). Thus, whether DmIKKε controls SOP cell development and the formation of macrochaetae was examined in vivo using DmIKKε IR transgenic flies. The knockdown of DmIKKε in the scutellum using the scabrous-GAL4 (Sca-GAL4) driver resulted in the appearance of extra macrochaetae. Two sets of Senseless-positive SOP cells were detected in a control wing disc; however, extra SOP cells were detected in wing discs expressing diap1 or DmIKKε IR. These observations suggest that DmIKKε may affect the caspase activity in proneural clusters to determine the correct number of SOP cells (Kuranaga, 2006).

The caspase activity in the proneural clusters of flies expressing SCAT3 under the Sca-GAL4 driver was determined. In the absence of cell-death stimuli, endogenous caspase activity was detected in the scabrous-expressing proneural clusters in wild-type wing discs; however, the expression of DIAP1 or the dominant-negative form of DRONC (Kanuka, 2005b) suppressed the caspase activity. Similarly, the knockdown of DmIKKε also effectively suppressed caspase activity in the scabrous-expressing proneural clusters of the wing discs. Thus, DmIKKε appears to determine the number of macrochaetae through the regulation of caspase activity in a nonapoptotic fashion (Kuranaga, 2006).

Ik2 is required for mRNA localization during oogenesis

In both Drosophila and mammals, IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. Mutations were identified in ik2, the gene that encodes one of two Drosophila IKKs; the gene is essential for viability. During oogenesis, ik2 is required in an NF-kappaB-independent process that is essential for the localization of oskar and gurken mRNAs; as a result, females that lack ik2 in the germline produce embryos that are both bicaudal and ventralized. The abnormal RNA localization in ik2 mutant oocytes can be attributed to defects in the organization of microtubule minus-ends. In addition, both mutant oocytes and mutant escaper adults have abnormalities in the organization of the actin cytoskeleton. These data suggest that this IkappaB kinase has an NF-kappaB-independent role in mRNA localization and helps to link microtubule minus-ends to the oocyte cortex, a novel function of the IKK family (Shapiro, 2006).

Protein kinases of the IkappaB kinase (IKK) family are known for their roles in innate immune response signaling pathways in both mammals and Drosophila. Mammalian IKKs all have roles in immune responses, but have a variety of targets. and IKKβ were identified in a protein complex that phosphorylates IkappaB and targets it for degradation, thereby allowing the nuclear localization and activation of NF-kappaB transcription factors. Gene targeting experiments in the mouse demonstrated that IKKβ, but not IKKalpha, is required for NF-kappaB activation by pro-inflammatory stimuli through receptors such as TLR4. IKKα activates the Rel/p52 transcription factor, because it activates proteolytic processing of the p100 precursor of p52 in an IkappaB-independent process. IKKepsilon and TANK binding kinase 1 (TBK1) are required to phosphorylate and activate the transcription factor Interferon regulatory factor 3 (IRF3) in response to viral infection. In addition to these immune response functions, IKKα has an NF-kappaB-independent role in epidermal differentiation and limb development (Shapiro, 2006 and references therein).

Dorsoventral patterning of the Drosophila embryo relies on the activation of Dorsal, a Rel-family transcription factor, by a signaling pathway that is homologous to mammalian TLR pathways. In response to activation of the receptor Toll, Cactus (the Drosophila IkappaB) is degraded, which allows Dorsal to move to embryonic nuclei and activate genes, such as twist, that are required for specification of ventral cell types. Phosphorylation of Cactus is required for its degradation, but the responsible kinase has not been identified. The Drosophila genome encodes two members of the IKK family. DmIkkβ (ird5 - FlyBase) is essential for the response to bacterial infection. DmIkkβ is required for proteolytic processing and activation of Relish, a p100-like Rel/ankyrin-repeat protein, like the role of mammalian IKKα in the activation of p100. The function of the second Drosophila protein kinase of the IKK family, ik2 (IkappaB kinase-like 2), has not been characterized, but it was a good candidate to control the phosphorylation and degradation of Cactus (Shapiro, 2006).

To test whether ik2 encodes a Cactus kinase, the phenotypes caused by loss of ik2 function were characterized. This study presents data showing that Ik2 is essential for dorsoventral and anteroposterior embryonic patterning of the Drosophila embryo. However, Ik2 does not act as a Cactus kinase, but exerts its effects on embryonic patterning through the localization of specific mRNAs during oogenesis. The data indicate that Drosophila Ik2 regulates RNA localization through regulation of the cytoskeleton and define a novel function for this protein family (Shapiro, 2006).

A saturation mutagenesis experiment had identified lethal complementation groups in polytene chromosome region 38E, the region that includes the ik2 gene. Missense mutations were identified in the ik2 kinase domain in all five alleles of the l(2)38Ea complementation group. A sixth allele, ik2^alice, identified in a genetic mosaic screen for maternal effect mutations, had a missense mutation in the C-terminal end of the kinase domain. All six ik2 alleles caused recessive lethality, and the majority of the mutants die as first instar larvae. At low temperature and in uncrowded culture conditions, rare escaper adults (<1%) were observed, but they died shortly after eclosion (Shapiro, 2006).

If ik2 encoded the Cactus kinase, embryos produced by females that lack ik2 would not be able to degrade Cactus, so Dorsal would not enter embryonic nuclei to activate genes required for ventral cell fate specification and the embryos would be dorsalized. Because ik2 mutations were lethal, FRT/FLP recombination combined with the ovo^D dominant female-sterile mutation were used to generate mutant clones in the female germline. More than 95% of the embryos laid by ik2^alice and ik2¹ mutant females did not hatch; however, larval cuticle preparations showed that none of the embryos were dorsalized. Instead, the majority of embryos produced by ik2^alice and ik2¹ mutants had a bicaudal phenotype, ranging from headless embryos to embryos with a duplicated abdomen in place of the head and thorax. In addition to this anteroposterior patterning defect, a large number of embryos from both ik2^alice and ik2¹ germline clones had expanded ventral cuticular structures, the opposite of the expected phenotype. Some embryos were both ventralized and bicaudal, with expanded ventral denticle bands and filzkörper (a posterior structure) in both the tail and the anterior of the embryo. Both ik2 alleles produced bicaudal and ventralized embryos, but 89% of the embryos produced by ik2^alice mutant females were bicaudal with no apparent dorsoventral abnormalities, whereas only 47% of embryos produced by ik2¹ mutant females were bicaudal, and the remainder of the embryos appeared to be too ventralized to score for ectopic posterior cuticular structures. A similar range of phenotypes was observed in embryos produced by ik2², ik2³ and ik2⁵ females with mutant germline clones (Shapiro, 2006).

Contrary to the prediction based on its sequence, ik2 does not act as a Cactus/IkappaB kinase in the Drosophila embryo. The embryonic ventralization caused by loss of ik2 is the opposite of the phenotype predicted for a Cactus kinase, and all effects of ik2 on the dorsoventral pattern of the embryo can be explained by a loss of activity of the Grk/Egfr pathway during oogenesis. Embryos that lack maternal activity of both Drosophila IKKs, Ik2 and DmIkkβ, are ventralized and are indistinguishable from ik2 single mutants, which rules out the possibility that the Drosophila IKKs act in both the Grk/Egfr and Toll pathways, and indicates that an unidentified kinase of another family is required to target Cactus for degradation. Additional experiments will be required to test whether ik2 plays other roles in the immune response (Shapiro, 2006).

It was found that instead of playing a role in Cactus degradation, Drosophila ik2 is required for the localization of specific mRNAs during oogenesis. Both the actin and microtubule cytoskeletons are disrupted in ik2 mutants, and defects in microtubule-based transport are sufficient to account for the defects in mRNA localization seen in ik2 mutants. Because Drosophila ik2 is specifically required for organization of the oocyte cytoskeleton, the results raise the possibility that some of the NF-kappaB-independent roles of the mammalian IKKs may act through the cytoskeleton (Shapiro, 2006).

The embryonic patterning defects caused by the loss of ik2 function are due to the failure to transport all osk mRNA to the posterior pole of ik2 mutant oocytes, which leads to bicaudal embryos, and failure to localize grk mRNA to the dorsal anterior of the oocyte, which leads to ventralized embryos. Loss of ik2 has a milder effect on bcd mRNA localization; bcd is correctly localized in most oocytes, but is not tightly restricted to the anterior pole in a minority of cases (Shapiro, 2006).

Many lines of evidence indicate that osk localization to the posterior pole depends on kinesin and gurken localization to the dorsoanterior corner depends on dynein. However, the kinesin and dynein motors in the oocyte are interdependent. For example, posterior localization of dynein and the anterodorsal localization of gurken are both disrupted in Khc mutants, and hypomorphic Dhc mutants have a reduced amount of Khc-β-gal at the posterior pole. Both motor systems are at least partially functional in ik2 mutants: most oskar is localized to the posterior pole of the oocyte (a kinesin-dependent process) and grk mRNA is localized anteriorly (a dynein-dependent process) (Shapiro, 2006).

Several lines of evidence suggest that the RNA localization defects seen in ik2 oocytes are associated with defects in a subset of dynein-mediated, minus-end-directed transport processes. The movement of grk mRNA to the dorsoanterior corner of the oocyte depends on two sequential dynein-based movements: grk mRNA moves first to the anterior of the oocyte along microtubules with plus-ends at the posterior pole and minus-ends at the anterior, and then moves dorsally on microtubules with minus-ends that form a cage around the oocyte nucleus. The dorsal movement of grk mRNA is specifically blocked in ik2 mutants, which would be consistent with a failure in this dynein-based movement. Restriction of bcd mRNA to the anterior margin of the oocyte, which is disrupted in some ik2 oocytes, depends on the swallow gene product, which binds dynein light chain. Overexpression of dynamitin disrupts dynein function and causes changes to the localization of grk and bcd mRNA that are similar to the phenotype of ik2 oocytes. In addition, BicD mutations produce a maternal effect phenotype similar to that of ik2. BicD is part of a protein complex with dynein light chain in early oocytes, neuroblasts and the early embryo, and has been proposed to link cargo to microtubules in both Drosophila and mammalian cells (Shapiro, 2006).

Although this evidence links ik2 to a dynein transport system, the most penetrant phenotype in ik2 mutants is osk mislocalization and subsequent production of bicaudal embryos, a kinesin-dependent process. However, loss of ik2 function, like the BicD gain-of-function mutations, does not eliminate kinesin function, because the majority of osk mRNA accumulates at the posterior pole. Because the kinesin and dynein motors in the oocyte are interdependent, osk mRNA mislocalization could be caused by a decreased kinesin activity that is secondary to dynein disruption (Shapiro, 2006).

In addition to defects in minus-end-directed transport, the organization of the microtubules is also perturbed in ik2 oocytes. The plus-ends of microtubules are localized correctly to the posterior pole of the oocyte. However, there are abnormal aggregates of microtubules around the oocyte nucleus, where a population of microtubule minus-ends is normally anchored, and the microtubule minus-end marker, Nod-β-gal, is not localized at the anterior of the oocyte. These defects suggest that abnormal organization of microtubule minus-ends during mid-oogenesis could be the basis of the defect in minus-end-directed transport (Shapiro, 2006).

The adult bristles and ovaries of ik2 mutants also displayed abnormalities in the actin cytoskeleton. The bristle defects are nearly identical to those caused by mutations in actin-associated proteins, or to bristles that were treated with F-actin-inhibitors. Bristles contain a central core of microtubules, but mutations in the dynein heavy chain gene Dhc64C or treatment with drugs that disrupt microtubule dynamics do not cause bristle phenotypes like the thick, branched bristles seen in ik2 mutants. The actin cytoskeleton of the oocyte is also disrupted in ik2 mutants, with ectopic sites of actin polymerization in the ooplasm. These actin defects are distinct from those caused by mutations that affect nurse cell ring canal actin, which suggests that actin organization is not globally disrupted in ik2 mutants and that the actin defects are restricted to the oocyte cortex (Shapiro, 2006).

Recent data have defined two sets of microtubules in the oocyte that are both nucleated from minus-ends at the centrosome associated with the oocyte nucleus; one set remains associated with the oocyte nucleus, whereas the remaining microtubules shift their minus-ends from the oocyte to the cortex. It was suggested that translocation of the minus-ends of the latter set of microtubules to the cortex could depend on actin and motor proteins. The current data suggest that anchoring of microtubule minus-ends to the oocyte cortex depends upon an Ik2-dependent interaction of microtubule minus-ends with the F-actin network, analogous to the interaction of microtubule plus-ends with the actin cytoskeleton through microtubule tip proteins (Shapiro, 2006).

The phenotypes of ik2 in the ovary and adult bristles are very similar to those caused by mutations in spn-F (Abdu, 2006). Like ik2 mutations, null mutations in spn-F affect the localization of osk and grk mRNAs during oogenesis, and cause bicaudal and ventralized embryos. spn-F mutant oocytes have ectopic sites of F-actin polymerization, and spn-F bristles are similar to ik2 mutant bristles. Ik2 and Spn-F have been shown to interact in a yeast two-hybrid screen, which suggests that these proteins can form a complex. Spn-F associates specifically with microtubule minus-ends (Abdu, 2006). It is therefore proposed that Ik2 and Spn-F act together to regulate interactions between the minus-ends of microtubules and the actin-rich cortex (Shapiro, 2006).

Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis

Pruning is a widely observed mechanism for developing nervous systems to refine their circuitry. During metamorphosis, certain Drosophila sensory neurons undergo large-scale dendrite pruning to remove their larval branches before regeneration of their adult dendrites. Dendrite pruning involves dendrite severing, followed with debris removal. Little is known about the molecular mechanisms underlying dendrite severing. This study shows that both the Ik2 kinase and Katanin p60-like 1 (Kat-60L1) of the Katanin family of microtubule severing proteins are required for dendrite severing. Mutant neurons with disrupted Ik2 function have diminished ability in severing their larval dendrites in pupae. Conversely, premature activation of Ik2 triggers precocious dendrite severing in larvae, revealing a critical role of Ik2 in initiating dendrite severing. A role was found for Kat-60L1 in facilitating dendrite severing by breaking microtubule in proximal dendrites, where the dendrites subsequently separate from the soma. This study thus implicates Ik2 and Kat-60L1 in dendrite severing that involves local microtubule disassembly (Lee, 2009).

Although the requirement of Ik2 signaling in dendrite severing may involve DIAP1 downregulation, inactivation of Ik2 function caused a much stronger dendrite severing phenotype than DIAP1 GOF mutation did, suggesting that Ik2 signals might be mediated by pathways other than or in addition to those involving DIAP1 and caspases to trigger dendrite severing in class IV da neurons during dendrite pruning. The function of ik2 is also required in several other developmental processes in Drosophila, including the regulation of F actin dynamics during trachea and bristle morphogenesis, and the organization of cytoskeletons for proper mRNA localization during oogenesis. One common feature in all these developmental processes is the dynamic organization of cytoskeletons. This study identifies one of the potential regulators of cytoskeletal rearrangement concomitant with dendrite pruning (Lee, 2009).

Microtubule disassembly is a cellular feature typically observed in developmental axon pruning, Wallerian degeneration, and some neurodegenerative diseases. During axon pruning of MB γ neurons in Drosophila, the disruption of microtubule cytoskeletons precedes the disruption of other cellular makers in axons. Targeted microtubule breakage in the proximal dendrites of class IV da neurons also takes place before the membrane separation from the soma. Moreover, the breakage of microtubule cytoskeletons in the proximal dendrites was suppressed in mutants with impaired ecdysone signaling, and in ik2 and kat-60L1 mutants (Lee, 2009).

In an RNAi screen for molecules that are responsible for local microtubule disruption in the proximal dendrites, three candidate genes encoding microtubule-severing proteins, kat-60, kat-60L1, and spastin, were tested and prominent dendrite severing phenotypes were found only in kat-60L1 RNAi mutants. The class IV da neurons in spastin LOF mutants did not show noticeable dendrite severing phenotypes. Owing to the lethality caused by kat-60 mutations and the proximity of kat-60 gene to the centromere prohibiting MARCM analyses, it has been difficult to examine the dendrite severing phenotype of class IV neurons in kat-60 mutants besides the RNAi mutants. The in situ hybridization signals of kat-60L1 mRNA in fly embryos reported by the Berkeley Drosophila Genome Project indicate that kat-60L1 is expressed in the embryonic nervous system, including brain and ventral nerve cord. The genetic study indicates that one of the Kat-60L1 functions in the nervous system is to efficiently sever the proximal dendrites of class IV neurons in pupae. Whether Kat-60 also contributes to dendrite severing remains to be determined (Lee, 2009).

The protein sequence of Kat-60L1 shows high homology to Drosophila Katanin-60. Katanin, a microtubule-severing protein, is a heterodimer composed of 2 subunits, p60 that possesses the microtubule-severing activity, and p80 that regulates the enzymatic activity and targeting of p60. Drosophila Kat-60 can interact with Drosophila Kat-80 in yeast 2-hybrid screen. The N-terminal 29 residues of human Katanin p60 required for interaction with human Katanin p80 is conserved in Drosophila Kat-60, but absent in Kat-60L1, indicating that Kat-60L1 is unlikely to be regulated by Kat-80 through similar direct interaction. It has been shown that phosphorylation could also regulate the microtubule-severing activity of Katanin during mitosis. Similar mechanisms could regulate the activity of Kat-60L1 to sever microtubules. Since both Ik2 kinase and Kat-60L1 are involved in dendrite severing of class IV da neurons, it will be of interest to test whether Ik2 kinase might regulate Kat-60L1 activity (Lee, 2009).

One intriguing question about the pruning in class IV da neurons concerns the mechanisms by which neurons restrict the pruning activity only to their dendrites. It is possible that some microtubule-associated proteins (MAP) may preferentially bind to the microtubules in dendrites or those in axons and thus provide differential protection against the microtubule-severing activity of Kat-60L1. For example, in cultured rat hippocampal neurons, the microtubules in dendrites are more susceptible to excess activity of overexpressed Katanin p60 than those in axons, and the axonal Tau proteins protect axonal microtubules from severing by Katanin. The differential binding preference for microtubules in dendrites versus axons could also arise from regional posttranslational modification of MAPs. For example, the phosphorylated MAP4, MAP2, and Tau display lower affinity to microtubules than their non-phosphorylated counterparts. It is also likely that the posttranslational modification of microtubule in dendrites is different from that in axons, since katanin has been suggested to display differential preference to microtubules with different modification in tetrahymena and in C. elegans. In future studies, it would be interesting to test these possibilities to delineate the mechanisms of dendrite-specific pruning (Lee, 2009).

REFERENCES

Search PubMed for articles about Drosophila I-kappaB kinase epsilon

Abdu, U., Bar, D. and Schupbach, T. (2006). spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila. Development 133: 1477-1484. PubMed ID: 16540510

Arama, E., Agapite, J. and Steller, H. (2003). Caspase activity and a specific cytochrome C are required for sperm differentiation in Drosophila. Dev. Cell 4(5): 687-97. Medline abstract: 12737804

Dubin-Bar, D., Bitan, A., Bakhrat, A., Kaiden-Hasson, R., Etzion, S., Shaanan, B. and Abdu, U. (2008). The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis. BMC Cell Biol 9: 51. PubMed ID: 18796167

Fernando, P., Brunette, S. and Megeney, L. A. (2005). Neural stem cell differentiation is dependent upon endogenous caspase 3 activity. FASEB J. 19(12): 1671-3. Medline abstract: 16103108

Geisbrecht. E. R. and Montell, D. J. (2004). A role for Drosophila IAP1-mediated caspase inhibition in Rac-dependent cell migration. Cell 118(1): 111-25. Medline abstract: 15242648

Hoopfer, E. D., Penton, A., Watts, R. J. and Luo, L. (2008). Genomic analysis of Drosophila neuronal remodeling: a role for the RNA-binding protein Boule as a negative regulator of axon pruning. J Neurosci 28: 6092-6103. PubMed ID: 18550751

Huang, J., Wu, S., Barrera, J., Matthews, K. and Pan, D. (2005). The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell 122(3): 421-34. Medline abstract: 16096061

Huh, J. R., et al. (2004). Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization. PLoS Biol. 2(1):E15. Medline abstract: 14737191

Kanuka, H., et al. (2005a). Gain-of-function screen identifies a role of the Sec61alpha translocon in Drosophila postmitotic neurotoxicity. Biochim. Biophys. Acta 1726(3): 225-37. Medline abstract: 16243437

Kanuka, H., et al. (2005b). Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in neural precursor development. EMBO J. 24(21): 3793-806. Medline abstract: 16222340

Kishore, N., et al. (2002). IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2. J. Biol. Chem. 277(16): 13840-7. Medline abstract: 11839743

Kuo, C. T., Jan, L. Y. and Jan, Y. N. (2005). Dendrite-specific remodeling of Drosophila sensory neurons requires matrix metalloproteases, ubiquitin-proteasome, and ecdysone signaling. Proc Natl Acad Sci U S A 102: 15230-15235. PubMed ID: 16210248

Kuranaga, E., Kanuka, H., Tonoki, A., Takemoto, K., Tomioka, T., Kobayashi, M., Hayashi, S. and Miura, M. (2006). Drosophila IKK-related kinase regulates nonapoptotic function of caspases via degradation of IAPs. Cell 126: 583-596. PubMed ID: 16887178

Lee, H. H., Jan, L. Y. and Jan, Y. N. (2009). Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis. Proc Natl Acad Sci U S A 106: 6363-6368. PubMed ID: 19329489

Lin, T., et al. (2015). Spindle-F is the central mediator of Ik2 kinase-dependent dendrite pruning in Drosophila sensory neurons. PLoS Genet 11: e1005642. PubMed ID: 26540204

Oshima, K., et al. (2006). IKK epsilon regulates F actin assembly and interacts with Drosophila IAP1 in cellular morphogenesis. Curr. Biol. 16(15): 1531-7. Medline abstract: 16887350

Otani, T., Oshima, K., Onishi, S., Takeda, M., Shinmyozu, K., Yonemura, S. and Hayashi, S. (2011). IKKepsilon regulates cell elongation through recycling endosome shuttling. Dev Cell 20: 219-232. PubMed ID: 21316589

Otani, T., Oshima, K., Kimpara, A., Takeda, M., Abdu, U. and Hayashi, S. (2015). A transport and retention mechanism for the sustained distal localization of Spn-F-IKKepsilon during Drosophila bristle elongation. Development 142: 2338-2351. PubMed ID: 26092846

Peters, R. T., Liao, S. M. and Maniatis, T. (2000). IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex. Mol. Cell 5(3): 513-22. Medline abstract: 10882136

Schwerk, C. and Schulze-Osthoff, K. (2003). Non-apoptotic functions of caspases in cellular proliferation and differentiation. Biochem. Pharmacol. 66(8): 1453-8. Medline abstract: 14555221

Shapiro, R. S. and Anderson, K. V. (2006). Drosophila Ik2, a member of the I kappa B kinase family, is required for mRNA localization during oogenesis. Development 133: 1467-1475. PubMed ID: 16540511

Tojima, Y., et al. (2000). NAK is an IkappaB kinase-activating kinase. Nature 404: 778-782. Medline abstract: 10783893

Williams, D. W. and Truman, J. W. (2005). Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons. Development 132: 3631-3642. PubMed ID: 16033801

date revised: 20 June 2010

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