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).

GENE STRUCTURE

cDNA clone length - 2744 (isoform A)

Bases in 5' UTR - 71

Exons - 6

Bases in 3' UTR - 537

PROTEIN STRUCTURE

Amino Acids - 711 (isoform A)

Structural Domains

A gene on the Drosophila second chromosome, ik2, was identified as a member of the IKK family. The closest mammalian homologs of Drosophila Ik2 are IKKepsilon and TANK binding kinase 1 (TBK1), which are 60%-61% identical to Ik2 in the kinase domain and 51% identical across the entire protein; by contrast, Ik2 is only 28% identical to the other Drosophila IKK, DmIkkβ (Shapiro, 2006).

CG2615 shows similarity to the IKK-related kinase family and is named DmIKKε (also known as Ik2). Each of the APTX7 alleles contained a G→A base change in the putative kinase domain of DmIKKε, thereby altering a Gly to an Arg at position 19 (APTX7⁴⁶), an Asp to an Asn at position 160 (APTX7⁶⁶), or a Gly to an Asp at position 250 (APTX7³⁶). DmIKKε most closely resembles the human NAK (also known as T2K or TBK1) and IKKε (also known as IKKι); BLAST searches of the fly genome failed to reveal a second IKKε-like gene, suggesting that DmIKKε may perform all of the functions that the two IKK-related kinases have in mammals (Kuranaga, 2006).

date revised: 20 April 2007

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