TGF-ß activated kinase 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation and Ectopic Expression | References
Gene name - TGF-ß activated kinase 1

Synonyms - dTAK1

Cytological map position - 19E1

Function - signaling

Keywords - immune response, JNK pathway, planar polarity, eye

Symbol - Tak1

FlyBase ID: FBgn0026323

Genetic map position -

Classification - serine/threonine protein kinase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |

Recent literature
West, R. J., Lu, Y., Marie, B., Gao, F. B. and Sweeney, S. T. (2015). Rab8, POSH, and TAK1 regulate synaptic growth in a Drosophila model of frontotemporal dementia. J Cell Biol 208: 931-947. PubMed ID: 25800055
Summary:
Mutations in genes essential for protein homeostasis have been identified in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients. Why mature neurons should be particularly sensitive to such perturbations is unclear. This study identified mutations in Rab8 in a genetic screen for enhancement of an FTD phenotype associated with ESCRT-III dysfunction. Examination of Rab8 mutants or motor neurons expressing a mutant ESCRT-III subunit, CHMP2B(Intron5), at the Drosophila melanogaster neuromuscular junction synapse revealed synaptic overgrowth and endosomal dysfunction. Expression of Rab8 rescued overgrowth phenotypes generated by CHMP2B(Intron5). In Rab8 mutant synapses, c-Jun N-terminal kinase (JNK)/activator protein-1 and TGF-beta signaling were overactivated and acted synergistically to potentiate synaptic growth. Novel roles were identified for endosomal JNK-scaffold POSH (Plenty-of-SH3s) and a JNK kinase kinase, TAK1, in regulating growth activation in Rab8 mutants. These data uncover Rab8, POSH, and TAK1 as regulators of synaptic growth responses and point to recycling endosome as a key compartment for synaptic growth regulation during neurodegenerative processes

Aggarwal, P., Gera, J., Mandal, L. and Mandal, S. (2016). The morphogen Decapentaplegic employs a two-tier mechanism to activate target retinal determining genes during ectopic eye formation in Drosophila. Sci Rep 6: 27270. PubMed ID: 27270790
Summary:
Understanding the role of morphogen in activating its target genes, otherwise epigenetically repressed, during change in cell fate specification is a very fascinating yet relatively unexplored domain. The in vivo loss-of-function genetic analyses in this study reveal that specifically during ectopic eye formation, the morphogen Decapentaplegic (Dpp), in conjunction with the canonical signaling responsible for transcriptional activation of retinal determining (RD) genes, triggers another signaling cascade. Involving dTak1 and JNK, this pathway down-regulates the expression of polycomb group of genes to do away with their repressive role on RD genes. Upon genetic inactivation of members of this newly identified pathway, the canonical Dpp signaling fails to trigger RD gene expression beyond a threshold, critical for ectopic photoreceptor differentiation. Moreover, the drop in ectopic RD gene expression and subsequent reduction in ectopic photoreceptor differentiation resulting from inactivation of dTak1 can be rescued by down-regulating the expression of polycomb group of genes. These results unravel an otherwise unknown role of morphogen in coordinating simultaneous transcriptional activation and de-repression of target genes implicating its importance in cellular plasticity. 


BIOLOGICAL OVERVIEW

Mammalian TAK1 was initially identified by a complementation assay in yeast for its ability to substitute for the MAPKKK Ste11p in the yeast MAPK pheromone pathway (Yamaguchi, 1995). TAK1 is a MAPKKK and contains a protein kinase domain in its N-terminal region that is ~30% identical to the catalytic domains of RAF1 and MEKK1. In mammals TAK1 can function in a signal transduction pathway that is triggered by the TGFß superfamily of ligands. In addition, the overexpression of Xenopus TAK1 and an upstream activator called TAB1 induce embryonic ventralization. Studies of dominant negative mutant forms of TAK1 place TAK1 activity downstream of the BMP2/4 receptors (Shibuya, 1998; Vidal, 2001 and references therein).

Besides TGFß signaling, TAK1 appears to be involved in various other signal transduction pathways. Cell culture experiments indicate that ceramide stimulates the TAK1 kinase activity and that ceramide-induced JNK/SAPK activation is blocked by a dominant negative TAK1 mutant (Shirakabe, 1997). Biochemical studies also show that MAPKK4 and MAPKK3/MAPKK6 can be phosphorylated by TAK1, suggesting that TAK1 can activate the JNK pathway and/or the P38 MAPK pathways (Moriguchi, 1996; Wang, 1997), and other experiments demonstrate interactions between TAK1 and the Wnt signaling pathway (Ishitani, 1999). Several studies have pointed toward a role for TAK1 in the IL1-R/NF-kappaB signaling pathway (Sakurai, 1998; Ninomiya-Tsuji, 1999; Sakurai, 1999). Dominant negative TAK1 mutants prevent NF-kappaB activation by IL1 or the TLRs (Ninomiya-Tsuji, 1999; Irie, 2000), and TAK1 appears to activate NF-kappaB by stimulating the IkappaB kinase complex (IKK) that induces IkappaB degradation by the proteasome (Ninomiya-Tsuji, 1999; Sakurai, 1999). Furthermore, TAK1 interacts with TRAF6, which is a downstream effector of the IL1 receptor, suggesting that TAK1 links TRAF6 to the NIK-IKK cascade in the IL1-R/TLR signaling pathways (Ninomiya-Tsuji, 1999; Vidal, 2001 and references therein).

Drosophila Tak1 is required for Relish cleavage and antibacterial immunity (Vidal, 2001). Ectopic activation of Tak1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by Tak1 signaling. Expression of a dominant negative form of Tak1 during embryonic development results in various embryonic cuticle defects including dorsal open phenotypes (Takatsu, 2000). Phenotypes generated by overexpressing a dominant negative form of Tak1 (Takatsu, 2000; Mihaly, 2001) are not observed in the Tak1-deficient mutants (Vidal, 2001), although the dominant negative Tak1 mutant does phenocopy the Tak1 mutant immune phenotype (Vidal, 2001).

Dorsal closure, taking place in mid-embryogenesis, describes the morphogenetic movements of the epidermis in order to replace the amnioserosa on the dorsal side of the embryo. This event is driven by the concerted spreading of epidermal cells towards the dorsal midline, where the two contralateral epidermal cell layers meet and remain connected. The JNK signaling module and nuclear targets of JNK, the AP-1 transcription factors dJun and dFos, control the process of dorsal closure. Uncompleted or failed dorsal closure is indicative of disrupted JNK signaling. Mutations in all known components of the JNK signaling pathway result in dorsal open embryos (Mihaly, 2001).

During dorsal closure, the expression of the dpp and puc genes in cells of the leading edge is controlled by the JNK kinase module and the AP-1 transcription factors Jun and Fos. Leading edge cells show loss of puc and dpp expression when deficient for JNK signaling. Conversely, constitutive activation of JNK signaling in the embryonic epidermis by expressing RacV12 or Dcdc42V12 (via the UAS/GAL4 system) induces the upregulation of dpp and puc (Mihaly, 2001 and references therein).

To address the question of whether Tak1 can activate and act through the JNK MAPK module, Tak1 was expressed under the control of the en-GAL4 and pnr-GAL4 drivers and the induction of dpp and puc were monitored in the epidermis of stage 12-15 embryos (pnr is strongly expressed in cells of and neighboring the leading edge). In wild-type, dpp is expressed in two lateral stripes along the Drosophila embryo, and the dorsal most stripe corresponds to the leading edge of the epidermis. Overexpression of Tak1 with either GAL4 driver causes ectopic upregulation of dpp, as monitored by RNA in situ hybridization. Similarly, the analysis of embryos carrying one copy of the pucE69 lacZ enhancer trap by beta-galactosidase activity staining shows a clear and robust ectopic puc expression when Tak1 is overexpressed. These patterns of dpp and puc activation by Tak1 are identical to those observed with activated Jun, suggesting that the effect is direct and mediated by the JNK signaling pathway (Mihaly, 2001).

In summary, these data indicate that overexpression of Tak1 in the embryonic ectoderm is sufficient to induce high-level expression of both known JNK target genes. As the same upregulation of puc and dpp is observed with JNKK/HepAct and Jun (a JNK activated transcription factor), the data strongly suggest that Tak1 acts through the JNK/Jun(AP-1) module in the context of dorsal closure (Mihaly, 2001).

In the absence of a Tak1 mutation, fly stocks carrying a dominant negative (DN) version of Tak1 (UAS-Tak1K46R or UAS-Tak1D159A) were generated to mimic the LOF situation. Embryonic expression of such kinase dead Tak1 proteins by using the 69B-Gal4, pnr-Gal4 or arm-Gal4 driver lines does not significantly affect the development of the embryos. Although various cuticle defects were detected, the penetrance of these phenotypes, including segmentation, head involution and dorsal closure defects, were below 3% and thus were considered insignificant (Mihaly, 2001).

The expression of DN proteins might induce unspecific pleiotropic effects by interfering with other kinases or signaling pathways, and/or only reduce the function of a given protein partially. Moreover, although kinase dead DN-Tak1 constructs worked in other tissues, the results with a DN-Tak1 are in contrast to the observation by Takatsu (2000) that reports 7% of embryos with dorsal closure phenotypes and an additional 25% displaying head involution defects. The experiments reported in this paper do not reveal any significant embryonic phenotypes. Thus, the double-stranded RNAi technique was employed to analyze a Tak1 LOF phenotype in the embryo. RNAi has been shown to effectively block the function of the gene of interest. In addition, it is strictly gene-specific for the RNA injected (Mihaly, 2001).

Injection of double-stranded Tak1 RNA into wild-type embryos results in a variety of embryonic defects. About 17.5% of the embryos exhibit an anterior open phenotype. However, dorsal closure defects are seen rarely in only about 3% of embryos, raising the question of whether Tak1 is required for dorsal closure as expected from the overexpression studies. Nevertheless, the anterior open phenotype appears to be significant since control injections of other double-stranded RNAs (dsRNAs), where the background of such phenotypes was only 1% (approximately) (Mihaly, 2001).

An explanation for the very low penetrance of the dorsal closure phenotype might be a general insensitivity to interfere with the process by means of RNAi. In order to address this issue, a control experiment was performed injecting D-jun (a well established factor required for dorsal closure) dsRNA into preblastoderm embryos. For D-jun injections, over 90% of the embryos display strong dorsal open cuticles, mimicking the D-jun LOF null phenotype. These results indicate that dorsal closure can be disrupted by RNAi-based methodology. Since Tak1 transcripts are abundant in preblastoderm embryos, determined by RNA in situ hybridization, a possible caveat to the RNAi Tak1 experiment could be maternally-derived protein that cannot be overcome by means of RNAi. However, this maternal product should still be inhibited by the overexpression of DN-Tak1, which was not seen (Mihaly, 2001).

Taken together, these DN-Tak1 overexpression and RNAi experiments suggest that Tak1 is not strictly required for dorsal closure and rather might serve an auxiliary role in the process. However, as this is in contrast to the results of Tak1 overexpression (when JNK target gene expression is upregulated very efficiently), it might be possible that Tak1 is (at least in part) redundant with another JNKKK during embryogenesis, and thus even a complete removal of it would be compensated by other kinases (Mihaly, 2001).

In contrast to the lack of a critical role for Tak1 in dorsal closure, Tak1 is required during morphogenetic changes and the fusion of the epithelial wing disc cell layers that takes place in thoracic closure, acting in the context of JNK signaling. JNK signaling is required in thoracic closure. The notum of the adult animal is formed by tissue of the two collateral wing imaginal discs, which undergo extensive morphogenetic rearrangements during metamorphosis. LOF in hep/JNKK and kayak/D-Fos results in aberrant wing disc morphogenesis and failure of wing disc fusion, giving rise to a thoracic cleft along the dorsal midline in the adult. To test whether Tak1 can also act in this context, DN (kinase dead) forms of Tak1 (UAS-Tak1K46R or UAS-Tak1D159A) were overexpressed with ap-GAL4 and pnr-GA66L4 in the thoracic parts of the wing discs. Examination of such flies shows incomplete closure of the thorax, giving rise to a mild thoracic cleft at the dorsal midline of the notum. Although this phenotype is relatively weak, it has a very high penetrance of 91% and is highly reminiscent of that observed in hypomorphic allelic combinations of either hep/JNKK or kay/d-fos. This is also in agreement with the phenotypic result of expressing Puckered, a negative regulator of JNK signaling at the time when wing disc fusion occurs. Puc overexpression driven by pnrGAL4 leads to the same phenotypic thorax cleft defects. These observations suggest that Tak1 is required during morphogenetic changes and the fusion of the epithelial wing disc cell layers, acting in the context of JNK signaling (Mihaly, 2001).

Overexpression of wild-type forms of Tak1 in eye imaginal discs results in defects in ommatidial planar polarity. The Drosophila JNK cascade has also been implicated in the establishment of correct ommatidial polarity in the compound eye where it acts downstream of Fz/Dsh. In the Drosophila eye, planar polarization is reflected in the mirror-symmetric arrangement of the ommatidial units relative to the dorso-ventral midline, the so-called equator. This pattern is generated early in the third instar imaginal disc, when immature ommatidia rotate 90° towards the equator. Subsequently, they adopt opposite chiral forms depending upon whether they lie dorsally or ventrally off the equator. Mutations in planar polarity genes like frizzled (fz) or dishevelled (dsh) result in the loss of mirror-image symmetry, with the ommatidia being misrotated and having acquired random chirality. Similarly, overactivation of any known component of the Fz/planar polarity pathway (including the JNK components) in both cells of the R3/R4 pairs leads to randomization of rotation and chirality causing a phenotype similar that of the LOF mutants (Mihaly, 2001).

An examination was carried out to see whether directed overexpression of Tak1 in the eye imaginal disc of third instar larvae (at the time of planar polarity Fz/JNK signaling) can interfere with the correct establishment of planar polarity. To this end UAS-Tak1 was expressed in photoreceptor precursors R3/R4 in the eye imaginal disc (under the sev-enhancer GAL4 driver: sev>Tak1). This type of overexpression creates specific eye planar polarity phenotypes with Fz, Dsh and other components of planar polarity signaling. Weak Tak1 expression (by rearing the flies at 18°C) causes a specific phenotype reminiscent of that caused by the components of planar polarity signaling, with polarity defects affecting both rotation and chirality, and also some loss of photoreceptors. This phenotype is already evident with the appropriate markers (e.g. svp-lacZ) at the time of planar polarity establishment in the third instar eye disc, indicating that it is a primary defect in polarity establishment, and not due to late differentiation defects (Mihaly, 2001).

The GOF sev>Tak1 phenotype provides a tool to test for genetic interactions with mutations in components of the Fz/planar polarity pathway and other signaling cascades. In such genetic interaction assays, it was found that reducing the dosage of the JNK signaling components (hep, bsk and D-jun) causes a strong suppression of the sev>Tak1 phenotype. These results are consistent with Tak1 acting upstream of the JNK module in polarity signaling, and support the notion that Tak1 can act generally upstream of JNK signaling (Mihaly, 2001).

Several other signaling pathways and kinases were tested for genetic interaction with sev>Tak1. Whereas no interaction with components of the Dpp signaling pathway was found, dominant suppression, comparable to that of the JNK components, was found with deficiencies removing the p38 kinases (p38a and p38b) and mutant alleles of nemo. These interactions suggest that the sev>Tak1 eye phenotype depends in part on the activities of these other MAPKs as well and is consistent with the previously reported tissue culture experiments. The observation that the sev>Tak1 phenotype is less sensitive to the dosage of msn/STE20 might indicate that msn is acting upstream of Tak1 (Mihaly, 2001).

It is concluded that during Drosophila development Tak1 can act as a component of the JNK signal transduction module. In addition, genetic interactions during eye development suggest that Tak1 can also activate other kinases, for example p38 type MAPKs and the Nemo kinase, in this context. Although TAK was initially identified as a TGF-beta activated kinase in mammalian cell culture systems (Yamaguchi, 1995), no evidence has been found by either phenotypic or genetic interaction analysis for Tak1 acting in a dpp/TGF-beta-dependent manner during Drosophila development (Mihaly, 2001).

Initially, the TAK subfamily of MAPKKK kinases has been identified as being activated by TGF-beta signaling (Yamaguchi, 1995). Subsequently, tissue culture experiments have shown that these kinases are able to activate both JNK and p38 MAPK pathways. These studies with Drosophila indicate that Tak1 can act in the context of the JNK cascade. This conclusion is based on several lines of evidence: (1) overexpression of Tak1 in embryos activates the expression of the JNK target genes dpp and puc; (2) expression of DN Tak1 with pnrGAL4 or apGAL4 leads to thorax closure defects, also a reported JNK LOF phenotype; (3) overexpression of Tak1 in third instar eye imaginal discs interferes with the correct establishment of ommatidial polarity, reminiscent of Hep, Bsk and Jun; (4) the GOF Tak1 planar polarity phenotype is suppressed by hep, bsk and jun LOF alleles. Taken together, these observations argue for Tak1 acting through the JNK cascade in Drosophila (Mihaly, 2001).

However, the analyses of the Tak1 LOF phenotypes generated by RNAi and DN isoforms during embryogenesis are in conflict with this conclusion and argue for a requirement that is in part redundant. The RNAi analysis has revealed predominantly anterior open embryos, and only very rarely the typical dorsal closure defects, normally seen in mutants of JNK signaling components. In addition, expression of a DN-Tak1 in the embryo in this study did not produce any dorsal closure phenotypes. Thus, although anterior open phenotypes can also be observed in mutants defective for JNK signal transduction (usually associated with hypomorphic alleles), the lack of clear dorsal closure defects suggests that Tak1 could be either partially redundant or plays a neglectable role in this JNK-mediated process. Taken together with the GOF produced by overexpression of Tak1, resulting in the robust activation of JNK target genes, these observations support the idea that Tak1 might act partially redundantly in this process during embryogenesis. Alternatively, the data can be viewed as Tak1 being able to generally act through the JNK signaling module wherever it is activated, leaving the question of the actual physiological role open. It is quite possible that Tak1 mediates JNK signaling only in a subset of the wide range of biological processes assigned so far to JNK signal transduction in Drosophila (Mihaly, 2001).

The studies reported by Takatsu (2000) found that Tak overexpression behind the MF can affect photoreceptor differentiation and causes cell death, but has otherwise no major effect on eye development (although mild defects in pigment and bristle cell differentiation can be revealed). In contrast, Tak1 overexpression with the sev-Gal4 driver severely interferes with the correct establishment of ommatidial polarity. The difference between these results can be explained by the different GAL4 drivers used. Whereas sevGAL4 is expressed at high levels early (and transiently) in the R3/R4 precursors, GMR-GAL4 (used by Takatsu) only affects later processes and can cause cell death. It is worth noting that the GMR-GAL4 driver does not cause planar polarity defects even with known planar polarity genes like fz and RhoA. The GMR-GAL4-driven expression starts in all cells behind the MF but early on levels are too low to cause defects. For example, GMR-driven expression of RhoA does not cause any appreciable defects in the third instar eye imaginal disc (when photoreceptors are recruited and polarity is established), although the corresponding adult eye is strongly reduced and malformed (Mihaly, 2001).

The sev>Tak1 eye phenotype is highly reminiscent of those caused by sev-Fz and sev-driven activated JNK pathway components. Moreover, since it can be suppressed by JNK LOF alleles, it supports a link between Tak1 and JNK and their role in regulating planar polarity in the eye downstream of Fz (Mihaly, 2001).

Genetic studies indicate that Tak1 can act through kinases other than JNK. Consistent with previous cell culture studies (Moriguchi, 1996), the p38 type MAP kinases also suppress the Tak1 GOF phenotype. This observation is also in line with the proposal that the Drosophila bsk/JNK and p38a and p38b kinases act as redundant elements in planar polarity signaling. The TAK MAPKKK family has also been implicated in activating a Nemo-like kinase (Nlk)/LIT-1 in cell culture and in C. elegans embryos (Ishitani, 1999; Meneghini, 1999 and Shin, 1999). Interestingly, nemo (the founding member of the Nlk subfamily) has been first described as a Drosophila planar polarity gene, affecting ommatidial rotation. Mutations in nemo strongly suppress the Tak1-induced eye polarity phenotype, raising the possibility that a TAK-Nemo/LIT-1 signaling cassette is evolutionarily conserved in worms, flies and vertebrates. Moreover, the existence of a Tak1-Nemo signal relay would suggest an interesting new link between the JNK signaling pathway and the Nemo kinases. The upstream activators and downstream targets of the latter and their role in planar polarity generation are unknown. In such a scenario Tak1 could be acting upstream at a branching point of the JNK and NLK signaling routes (Mihaly, 2001).

Strikingly, the TAK function mediated by Nemo-like kinases in vertebrate cell culture studies and C. elegans appears to antagonize canonical Wnt signaling at the level of Arm/TCF. As the canonical Wnt pathway and Fz/planar polarity signaling split at the level of Dsh, it is interesting to note that they might again intersect further downstream to reinforce the difference in signaling by antagonizing the other signaling branch. Further insight into the TAK-Nemo link will help to clarify the pathway specificity (Mihaly, 2001).

Several studies have demonstrated an involvement of the JNK pathway in stress-induced apoptosis in vertebrate cells. The Drosophila JNK pathway has also been implicated in regulating apoptosis upon deregulation of dpp and wg signaling in the developing wing disc. Tissue culture and Xenopus embryo injection experiments have shown that members of the TAK family of MAPKKKs induce apoptosis when overexpressed. Consistent with this, Tak1 overexpression in imaginal discs also induces apoptosis. Is the Tak1-induced apoptosis mediated through the JNK pathway? There are at least two observations supporting this possibility: (1) overexpression of a constitutively activated form of Hep (HepAct) or Jun (JunAsp) can lead to a qualitatively very similar phenotype as Tak1 overexpression with reduced eye size; (2) the apoptosis phenotype generated by Tak1 overexpression is strongly suppressed by hep, bsk and jun LOF alleles. Thus, a TAK-JNK signaling module might control apoptosis not only in vertebrates, but also in flies. It remains unclear whether the overexpression of Tak1 and high JNK activation directly activates the apoptotic pathway or whether cell death is a secondary consequence of it (Mihaly, 2001).

In summary, it has been demonstrated that the Drosophila TAK ortholog Tak1 can act as a JNKKK in the context of all described JNK-mediated morphogenetic processes during fly development, as well as during JNK-induced apoptotic cell death. However, the lack of a statistically significant dorsal closure phenotype in the embryo suggests that it is not the only JNKKK involved in this context (Mihaly, 2001).

Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways

Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, this study demonstrates that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals is rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. The data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression (Delaney, 2006).

Innate immune responses are critical for a rapid host defense against pathogens. The signaling pathways that control these responses are present in all multicellular organisms, ranging from humans to flies, and are remarkably well conserved. Although the innate response lacks the antigen recognition capacity of vertebrate adaptive immunity, it is nevertheless complex and crucial for host survival. Drosophila is a proven genetic model organism for the study of innate immunity and has provided invaluable insights into the control of responses to infection (Delaney, 2006).

Toll and Imd are the founding members of two principal innate immune response signaling pathways in Drosophila. Toll signals through two NF-kappaB/Rel family transcription factors, Dif and Dorsal, and is required for responses to fungal and Gram+ bacterial infections. Imd signaling controls primarily Gram- bacteria-specific responses through the cleavage and activation of a third Rel family transcription factor, Relish, by the Drosophila caspase Dredd. Relish activation also requires an IkappaB kinase (IKK) complex that is itself activated by Imd signaling. The transcriptional targets of Dif and Relish are not entirely distinct. For example, cecropinA expression requires either Relish or Dif, or both, depending on the type and strain of infecting microorganism. More than 20 Drosophila genes have been implicated in these signaling pathways and nearly all of them have mammalian homologues with conserved immune functions (Delaney, 2006 and references therein).

Jun N-terminal kinase (JNK) signaling has been linked to stress responses, cell migration, apoptosis, and immune responses in both insects and mammals. JNK activity can be induced by infection, lipopolysaccharide, and inflammatory cytokines such as tumor necrosis factor (TNF) in flies and mammals. Null mutations in JNK signaling components are typically embryonic lethal in flies and thus unlikely to appear as targets of mutagenesis screens designed to detect immune response genes in living animals. An exception to this rule is dTAK1. Overexpression and dominant-negative studies indicated that dTAK1 can act as a JNK kinase kinase (Delaney, 2006 and references therein).

Previously characterized dTAK1 mutations, however, showed no apparent JNK-like phenotype, but failed to express Relish-dependent antimicrobial peptides, suggesting a role in the Imd pathway (Vidal, 2001). Previous epistasis analysis using the UAS/GAL4 overexpression system to ectopically express dTAK1 placed dTAK1 downstream of imd and upstream of the IKK complex in the Relish signaling pathway (Vidal, 2001). In vitro experiments implicated dTAK1 in the IKK-dependent phosphorylation of Relish in S2 cells (Delaney, 2006).

Evidence has been uncovered for a Relish-independent function of dTAK1 in the control of antimicrobial peptide gene expression. Several aspects of Relish activation appeared normal in infected dTAK1 mutant animals, including cleavage, nuclear localization, and promoter binding. Therefore whether JNK pathway components mediate dTAK1 function in the immune response was examined. Several lines of evidence are reported for dTAK1 acting through the JNK cascade in the innate immune response. First, overexpression of Hemipterous, a JNKK, rescued attacin and diptericin expression in dTAK1 mutant animals, whereas overexpression of the downstream Imd component Dredd did not. Second, it was found that expression of the Puckered (Puc) phosphatase, an inhibitor of JNK activity, suppressed the expression of antimicrobial peptide genes. To directly test for a JNK requirement in immune signaling, JNK mutant clones were induced in the fat body of larvae. Strikingly, diptericin, attacin, Metchnikowin, and Drosomycin expression was lost in the mutant tissue (Delaney, 2006).

It is concluded that the JNK pathway is required to mediate dTAK1 signaling during the Drosophila immune response. Furthermore, a model is proposed where the JNK and NF-kappaB signaling are both required to activate antimicrobial peptide gene expression during the immune response in the Drosophila fat body (Delaney, 2006).

The function of TAK1 in vertebrates has remained enigmatic. It was originally identified as a TGFβ-activated kinase, hence the name, in mammalian cell culture assays. However, follow-up work in multicellular contexts and in vivo analyses in vertebrates, C. elegans, and Drosophila have shown no clear link to TGFβ signaling, but rather suggest a role for TAK family kinases in JNK activation or as upstream activators of Nemo-like kinases. In mammalian systems, TAK1 is one of a number of kinases that can activate IKK complexes and, consequently, NF-kappaB signaling in vitro. In vitro studies of human cells have shown that targeting of TAK1 by RNAi reduces NF-kappaB activation by TNFalpha and IL-1 stimulation. Recent studies using fibroblasts derived from TAK1 mutant mouse embryos and mice with a B-cell-specific deletion of TAK1 showed that JNK activation was impaired in response to all stimuli tested in TAK1 mutant cells. Although NF-kappaB activation was impaired in response to stimulation by IL-1β, TNF, and TLR3 and TLR4 ligands, NF-kappaB activation by B-cell receptor or LT-β stimulation remained intact, suggesting a specific role for TAK1 upstream of IKKβ and JNK, but not IKKalpha. Interestingly, IKKalpha activation leads to the phosphorylation and processing of NF-kappaB2 from the p100 to the active p52 form, reminiscent of Relish activation in Drosophila (Delaney, 2006 and references therein).

Biochemical analyses in mammalian systems have demonstrated that TAK1 functions in multimeric protein complexes that can include TAB1, TAB2, and different TRAF proteins. The exact composition of these complexes seems to determine TAK1 responsiveness and downstream effects. In the fly, genetic studies found an interaction between dTRAF1 and dTAK1 in the activation of JNK signaling and apoptosis. Gain- and loss-of-function analyses indicate that dTRAF2, but not dTRAF1, is necessary for the activation of Relish-dependent gene expression; however, no interaction between dTRAF2 and dTAK1 in the activation of antimicrobial peptides has been reported (Delaney, 2006).

Genome-wide analyses that examined in vivo responses in Drosophila identified dJun and puc as genes potentially regulated by Toll and Imd signaling, suggesting a cross-regulation between these pathways and the JNK signaling pathway. A study recently reported that RNAi knockdown of kayak, msn, hep, or aop blocked E. coli-induced attacin and drosomycin expression in S2 cells. Furthermore, in related studies, it was also observed that, although dTAK1 RNAi-treated S2 cells failed to express an attacin reporter gene, Relish cleavage and nuclear localization remain intact in these cells. Other RNAi analyses in S2 cells have concluded that JNK signaling does not have a significant role in antimicrobial peptide gene expression. However, RNAi against hep or bsk seemed to partially block antimicrobial peptide induction, especially of attacin and cecropinA and, accordingly, attacinD levels were lower in microarrays when the JNK pathway was blocked. The current results confirm a positive role for JNK signaling in the antimicrobial peptide response in vivo (Delaney, 2006).

The placement of dTAK1 function upstream of JNK, rather than IKK, requires a remodeling of the signaling pathways that activate the antimicrobial peptide genes. Earlier models were based on studies that showed that dTAK1 mutations blocked the constitutive activation of diptericin by Imd overexpression (Vidal, 2001). In turn, IKK mutations blocked dTAK1-induced diptericin expression. One interpretation of these data places IKK directly downstream of dTAK1. However, if the activation of both JNK and IKK signaling pathways is required, then a disruption in either branch would be sufficient to suppress any upstream activation (Delaney, 2006).

Overexpression of dTAK1 is sufficient to induce antimicrobial peptide expression (Vidal, 2001). However, dTAK1 is an extremely potent activator of JNK signaling and apoptosis, and overexpression of dTAK1 could activate proteins that are not normal phosphorylation targets. Based on RNAi studies in S2 cells, dTAK1 is required for dIKK complex-dependent phosphorylation of Relish in vitro. This could reflect a stringent requirement for dTAK1 in blood cell-derived S2 cells that is different in fat body tissue (Delaney, 2006).

The new model would predict that overexpression of the Dredd caspase would be insufficient to activate fully the antimicrobial peptides in dTAK1 mutant animals and this is indeed the case. Overexpression of Dredd may be sufficient to induce antimicrobial peptide gene expression in a wild-type background because of inadvertent JNK pathway activation by ectopic caspase activity or by the heat-shock protocol itself. Alternatively, an additional role for Dredd has been proposed in the ubiquitin-mediated activation of dTAK1 and the dIKK complex (Zhou, 2005). The suppression by dTAK1 mutants of ectopic Dredd expression is consistent with this model as well, and does not distinguish between the two potential functions of Dredd. The current data are consistent with a model that places dTAK1 activity in a pathway parallel to the functions of IKK and Relish and in which both these pathways are required for the activation of antibacterial peptide genes such as diptericin and attacin (Delaney, 2006).

Promoter analyses of most antimicrobial peptide genes have not revealed any obvious binding sites for activator protein-1 (AP-1) complexes, the Jun/Fos heterodimer, and transcriptional mediator of JNK signaling. However, AP-1 binding sites can be quite diverse and are not always predictable directly from DNA sequence. Nevertheless, a recent study identified a functional AP-1 binding site in the attacinA promoter. These data suggest that AP-1 binding represses attacinA transcription by recruiting histone deacetylase 1 (dHDAC1) to the promoter. In contrast, in mammalian studies, c-Jun function is itself repressed by association with HDAC3. This repression is relieved upon JNK signaling. A similar mechanism may be employed in the Drosophila fat body. Accordingly, the sustained expression of attacin and other antimicrobial peptide genes in vivo would require an activation (or de-repression) of AP-1 function at the onset of the immune response. Such positive cooperation between AP-1 and NF-kappaB transcription factors was also seen in molecular studies of the human β-defensin-2 promoter (Delaney, 2006).

AP-1-dependent gene expression is normally rapid. Thus, if AP-1 activity is not directly required for diptericin expression, it could act indirectly through the activation of other genes. Alternatively, JNK could phosphorylate some targets other than the AP-1 complex proteins Jun and Fos. In mammalian studies, it has been shown that JNK can phosphorylate, and thereby inhibit, Insulin Receptor Substrate-1. However, the recent finding that RNAi against kayak/dFos can block antimicrobial peptide expression and the current dJun loss-of-function studies in vivo suggest that JNK does indeed signal through AP-1 to control expression of these genes (Delaney, 2006).

It is intriguing that overexpressed Puc not only blocks Relish-dependent antimicrobial peptide gene expression, but it also strongly blocks drosomycin expression, which is not true in dTAK1 mutants. This suggests that JNK or JNK-related proteins, for example, p38a, p38b, and MPK2, may also be important for other aspects of the immune response, for example, the Toll/Dif-dependent antimicrobial genes. The clonal analysis of JNK mutant tissue confirms that JNK is required not only for the expression of Gram--specific peptides diptericin and attacin, but also for Metchnikowin (Gram+/fungal specific) and drosomycin (fungal specific). Mutations in dTAK1 had less of an impact on Metchnikowin or drosomycin expression than on attacin, for example. Furthermore, reduced dJun activation occurred in dTAK1 mutant animals, indicating that other upstream kinases may be involved in the control of these genes. JNK is a member of a large family of mitogen-activated protein kinases (MAPKs). In the fly, there are at least five MAPKKKs, four MAPKKs, and five MAPKs, and so the potential redundancies are many. If these other proteins contribute to the immune response, how they do so has yet to be tested in genetic loss-of-function in vivo studies in the fat body (Delaney, 2006).

How JNK and NF-kappaB signals integrate to positively control gene expression is a critical question. This study has demonstrated that both are required for the expression of a particular set of immune responsive genes in vivo. Through the use of Drosophila genetics, it should be possible to identify novel immune response genes that are controlled cooperatively by JNK and NF-kappaB signaling. From promoter analysis of these genes, it may be possible to predict additional genes that are important for other biological processes. Both the JNK and NF-kappaB signaling pathways have been implicated many times in many different contexts. Continued analysis in Drosophila may lead to a general understanding of their roles in normal biological processes and developmental malignancies (Delaney, 2006).

Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing

Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila, the Imd pathway detects gram-negative bacterial infections through recognition of DAP-type peptidoglycan and activation of the NF-kappaB precursor Relish, which drives robust antimicrobial peptide (AMP) gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and K63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. This study demonstrates that Imd is rapidly K63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 (Effete) and Ubc13 (Bendless)-Uev1a, in conjunction with the E3 ligase Diap2. K63-ubiquitination activates the TGFβ-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of K63-chains and the addition of K48-polyubiquitin. This ubiquitin editing process results in the proteosomal degradation of Imd, which is proposed to function to restore homeostasis to the Drosophila immune response (Chen, 2017).

Previous work has demonstrated that Imd is cleaved, K63-polyubiquitinated and phosphorylated upon immune stimulation (Paquette, 2010). While this earlier study did not find K48- polyubiquitin chains, others have reported evidence of both K63- and K48-Imd modifications (Thevenon, 2009). However, the overall dynamics of and interconnections between these IMD post-translational modifications remained unclear. This study showed that peptidoglycan (PGN) from bacterial cell walls stimulation of S2 cells leads to five different Imd modifications: proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination, which leads to degradation of Imd through proteasome. These immune triggered signaling events are robust and incredibly rapid, with Imd cleavage and K63-polyubiquitination occurring as early as 2 minutes after PGN stimulation. While K63-modification peaks early and then steadily declines, K48-conjugation appears later, along with phosphorylation, and declines in proteasome-dependent manner. These kinetics argue that Imd is sequentially conjugated with K63 then K48 ubiquitin, so-called ubiquitin editing, as has been reported for IRAK1 and RIP1 in mammalian innate immune signaling pathways (Chen, 2017).

In addition to ubiquitination, two slow-migrating species of Imd were detected and shown to be phosphorylated forms. Judging by their size, these two phospho-forms appear to be derived from either full-length Imd (upper) or cleaved Imd (lower). Interestingly, persistence of phosphorylated Imd was observed in the proteasome-inhibited samples, suggesting that Imd is both K48-polyubiquitinated and phosphorylated before entering proteasome. Tak1 is required for these phosphorylation events as well as for ubiquitin editing, demonstrating key role for this MAP3K in a negative feedback loop (Chen, 2017).

Conjugation of ubiquitin usually occurs on lysine side chains of target proteins, and mass spectrometry of immuno-purified endogenously expressed Imd identified K137 and K153 as the sites of ubiquitin linkage. Note, the mass spec analysis includes 50% coverage of Imd, and a cluster of four lysine residues at the very C-terminus were not observed. Substitution of K137 and K153 residue with Arginine prevented signal- induced ubiquitination of Imd in S2 cells and reduced expression of the AMP gene Diptericin in both cells and flies. These results demonstrate that both lysine residues are required for K63- polyubiquitination and downstream signaling events. In S2 cells, mutation of single lysine residue led to a partial reduction of K63- ubiquitination and a partial reduction of AMP gene induction. Surprisingly, single lysine mutation did not correspondingly reduce Imd K48- polyubiquitination, while the double lysine mutation completely blocked it. These results suggest that even the reduced signal, mediated by a single K63-chain, is sufficient to trigger a robust feedback response with K48-chain formation. On the other hand, a complete block in K63-chains prevents Tak1 activation, which in turn fails to promote the ubiquitin editing of Imd. These findings are consistent with results observed with knockdown of Ubc5, Ubc13 and Uev1a. Ubc5 depletion prevented all K63 ubiquitination and signaling (as measured by Diptericin induction), and subsequent K48 modification was absent, while the Ubc13 and/or Uev1a knockdown showed residual K63 chains and greatly reduced Diptericin expression but robust K48- ubiquitination. These results also suggest that K48-polyubiquitination may occur on lysine residues beyond K137 and K153, although more detailed mass spectrometric analyses is required to map these sites more thoroughly (Chen, 2017).

On the other hand, only double lysine mutation leads to significant reduction of AMP expression in adult flies, and this is reduction is not as robust as in cultured cells. This pattern suggests that activation of the NF-κB protein Relish and its transcriptional targets do not solely rely on ubiquitination of Imd K137/153. Other possible ubiquitination targets include the upstream caspase Dredd, which has been shown to be critical for signaling (26), or the E3 ligase Diap2. This redundancy may represent multiple parallel mechanisms that contribute to the NF-κB activation. Furthermore, tissue specific immune differences may contribute to the discrepancy in Diptericin induction between macrophage-derived S2 cells and whole flies, in which the fat body rather than hemocytes is the major organ for inducible expression of AMPs (Chen, 2017).

Previous work has suggested that Diap2 is the E3 ligase for Imd ubiquitination. With the advantage of ubiquitin linkage specific antibodies, data presented in this study show that Diap2 is required for Imd K63-polyubiquitination and signaling, as measured by induction level of Diptericin. Moreover, the accumulation of cleaved but non-ubiquitinated Imd, in the Diap2 depleted cells and flies, provides further evidence that ubiquitination is downstream of Imd cleavage and highlights the role of Diap2 as the critical E3 in the K63- modification of Imd. In addition, Imd is no longer K48-modified when Diap2 is removed, suggesting that either Diap2 is also involved in K48- conjugation, or the failure of K63- polyubiquitination leads to the loss of K48- polyubiquitination. Given the role of Tak1 in this ubiquitin editing event, the latter hypothesis is favored, and another E3 is likely involved in the K48 conjugation (Chen, 2017).

In addition to E3 ligases, E2 ubiquitin conjugating enzymes are the other key factors in the ubiquitin conjugation reaction. Previously work has shown that Ubc5 and Ubc13-Uev1a are all involved in Imd ubiquitination. However, the mechanism by which these E2s collaborated was unclear. Results from in vitro reconstituted ubiquitination assays suggested a two-step reaction model for ubiquitin conjugation with different E2s. In particular, it was shown that some E2s, such as Ubc5, are effective at the initial ubiquitination of substrates but are ineffective at generating long chains, while other E2s, like Ubc13/Uev1a, are efficient at generating long ubiquitin chains but fail to conjugate substrate proteins. Thus, these two types of E2s can work together to generate long ubiquitin chains conjugated to target proteins. It is proposed that Imd undergoes ubiquitin chain initiation and elongation catalyzed by two separate E2s. Once cleaved, Imd interacts with Diap2 through its BIR repeats and is first modified by Ubc5-mediated substrate ubiquitination on lysines 137 and K153. Subsequently, the E2 complex of Ubc13-Uev1a pairs with an E3 (possibly Diap2 although another unidentifed E3 is not excluded) and switches the reaction to chain elongation mode, during which additional ubiquitin molecules are attached to the substrate-linked ubiquitin in a K63-specific manner. In the absence of Ubc13/Uev1a, Ubc5 alone is still able to elongate the polyubiquitin chains, but less efficiently and with unknown linkages (Chen, 2017).

Induction of Diptericin expression generally tracks with the K63-polyubiquitination signal (but not the total Ub signal) observed in various E2 knockdown cells. The one exception is the samples in which Ubc13 and Uev1a are both knocked down, and Ubc5 is still available. These samples display a similar K63 intensity as the single Ubc13 or Uev1a RNAi lanes, but Diptericin induction is lower, close to background levels. Ubc5 alone is known to conjugate ubiquitin without linkage specificity, a random polyubiquitin chain that consist of all seven types lysine linkage. Since the K63 antibody recognizes K63-linked diubiquitin, it is possible that Ubc5-mediated random ubiquitin chain elongation generates some K63 di-ubiquitin linkages which are detected by this antibody, but are unable support signaling due to their altered topology and limited amounts of K63-linkages. More detailed biochemical characterization of these Ubc5-catalyzed chains is required to confirm this hypothesis (Chen, 2017).

K48 modification of Imd shows a subtle difference relative to the K63 chains. Again, knock down of Ubc5 causes complete blocking of K48 conjugation, but depletion of Ubc13/Uev1a has no effect. The failure of K48 modification in the Ubc5 depleted cells may have two possible underlying causes. Firstly, the complete lack of K63 chains will fail to activate Tak1 and the subsequent ubiquitin editing feedback loop, while the Ubc13 and/or Uev1a RNAi display some residual K63 activity and thus can trigger Tak1 and the feedback response. Alternatively, Ubc5 might be directly required for Imd K48-polyubiquitination as shown in the degradation of proteins in multiple Drosophila pathways including eye development, maintenance of germline stem cells and apoptosis. These are not mutually exclusive possibilities (Chen, 2017).

Phosphorylation of Imd appears to be a major regulator of these ubiquitin-editing events. Knockdown of Tak1 prevents Imd phosphorylation in S2 cells and in adult flies. Moreover, immune-purified Tak1 can directly phosphorylate recombinant Imd in vitro, while neither JNKK nor IKK are required for phosphorylation of cleaved Imd, strongly arguing that Tak1 directly modifies Imd. RNAi-depletion or drug inhibition of Tak1 prevents K63-deubiquitination and the subsequent K48-polyubiquitination/proteasome-mediated degradation, leading to accumulation of cleaved but unphosphorylated Imd, presumably an intermediate during chain editing. From these results, it is inferred that Tak1-mediated phosphorylation of Imd is required for ubiquitin editing. Future studies will reveal the underlying mechanisms by which phosphorylation triggers K63-deubiquitination and K48 chain conjugation. Nonetheless, these results are consistent with earlier reports of Imd regulation by K63-deubiquitination and degradation (Chen, 2017).

Considering the results presented in this studdy together with earlier studies, the following model of Imd signal activation and subsequent down-regulation is proposed. One of the earliest events after PGN-stimulation is the rapid cleavage of Imd by the caspase-8 homolog DREDD at D30. Cleaved Imd then interacts with BIR-repeats of the E3 ligase Diap2 and is K63-polyubiquitinated through the sequential action of Ubc5, for substrate conjugation, and Ubc13-Uev1a, for catalyzing long K63 chains. These K63-polyubiquitin chains are then likely to activate the Tak1/Tab2 kinase complex through the conserved K63-binding motif in Tab2, which in turns signals through IKK complex to activate the NF-κB precursor Relish. Relish is central for the robust induction of AMP gene transcription. Meanwhile, Tak1 also mediates a retrograde signal that phosphorylates Imd and triggers ubiquitin editing, and leads to the degradation of Imd through proteasome. This regulatory interaction between Tak1 and Imd represents a novel homeostatic loop whereby the Drosophila immune response is rapidly activated but also quickly shutdown. Future studies are necessary to determine function of this feedback loop relative to other feedback mechanisms reported for the Imd pathway (Chen, 2017).


GENE STRUCTURE

cDNA clone length - 3349

Bases in 5' UTR - 915

Exons - 8

Bases in 3' UTR - 397


PROTEIN STRUCTURE

Amino Acids - 678

Structural Domains

The overall Drosophila Tak1 protein is similar to the vertebrate TAK1s, containing an NH2-terminal protein kinase domain as well as a long COOH-terminal domain. The kinase domain shows 56% identity and 73% similarity with the amino acid sequences of mTAK1. Phylogenetic analysis of the catalytic domain of Drosophila Tak1 with those of other Drosophila and vertebrate MAPKKK proteins indicate that its closest relatives are the vertebrate TAK1s. In the COOH-terminal region, Drosophila Tak1 is less well conserved with its vertebrate homologs. However, there is a stretch of 65 amino acids that is relatively well conserved between Drosophila and vertebrates (37% identity and 57% similarity to mTAK1). Interestingly, this region is almost completely missing in one of the alternative spliced forms of the human TAK1 (hTAK1c) (Takatsu, 2000 and references therein).


TGF-ß activated kinase 1: Evolutionary Homologs | Developmental Biology | Effects of Mutation and Ectopic Expression | References

date revised: 20 August 2001

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