BIOLOGICAL OVERVIEW

Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated to be an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and Drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).

Members of the tumor necrosis factor receptor (TNFR) superfamily can induce a wide spectrum of cellular responses, including cell proliferation, apoptosis, and differentiation. Most of these functions are mediated by a family of intracellular TNFR-binding proteins, the TNFR-associated factors (TRAFs). In humans and mice, the TRAF family consists of six members (TRAF1 to TRAF6): these proteins have a conserved stretch of amino acids near their C termini termed the TRAF domain. The TRAF domain is required for binding of these signal-transducing adaptor proteins to TNFRs. Two additional functional domains, the zinc finger domain and the RING finger domain, are located at the N terminus of TRAF protein and are proposed to be essential for the activation of specific downstream signaling components (Cha, 2003).

The involvement of TRAF family proteins in a variety of signal transduction pathways and cellular responses has been extensively studied by numerous cell culture-based studies. Mammalian TRAF2 and TRAF6 have been found to regulate the transcription of downstream target genes through the activation of two different intracellular signaling pathways, c-Jun N-terminal kinase (JNK) and nuclear factor-kappaB (NF-kappaB) signaling pathways. Even though many attempts have been made to distinguish the major TRAF-mediated signaling pathways and to deduce the in vivo function of each TRAF, these have been hampered by highly redundant roles of mammalian TRAFs in correlation with their signaling mechanisms (Cha, 2003 and references therein).

In addition to the intensive studies of TRAFs in the mammalian system, there were some pioneering studies to reveal the function of TRAFs in Drosophila melanogaster. Two Drosophila homologs of mammalian TRAFs, Traf1 and Traf2, have been identified, and the biochemical and cell culture-based studies with these proteins have shown that TRAF-dependent signaling pathways are indeed highly conserved in Drosophila. Traf2, like mammalian TRAF6, interacts with Drosophila ECSIT and Pelle, and consequently activates NF-kappaB in Schneider cells (Kopp, 1999). Traf1 interacts with Drosophila Ste20 kinase (Misshapen, msn) and induces a synergistic activation of JNK in mammalian cultured cells (Liu, 1999). However, a contradictory report shows the functional interactions between Traf1 and the NF-kappaB signaling pathway in cell culture-based experiments (Zapata, 2000). Despite these efforts, in vivo studies with a whole animal to confirm these in vitro experiments and to further dissect the specific signaling mechanisms of TRAFs regulating developmental and immunological functions remain to be accomplished (Cha, 2003).

To better understand the in vivo functions of DTRAFs, it is necessary to conduct genetic studies with various TRAF mutants. Fortunately, only two TRAFs exist in the Drosophila genome; this provides a lower number of the signaling molecules and more simple phenotypes and mutants to investigate (Grech, 2000, Kopp, 1999, Liu, 1999; Zapata, 2000). Using various convenient genetic systems, it has been possible to analyze the downstream signaling pathways of TRAFs under well-defined and physiologically relevant environments in Drosophila. Moreover, since the two major downstream signaling pathways for TRAFs, the mitogen-activated protein (MAP) kinase signaling cascades and the NF-kappaB pathway, are highly conserved between vertebrates and Drosophila, the genetic interactions between the DTRAFs and these downstream components can easily be confirmed in the fruit fly and applied to mammalian systems (Cha, 2003).

The downstream signaling pathways and physiological functions of Drosophila Traf1 and Traf2 have been analyzed by using their gain-of-function and loss-of-function mutants. The results indicate that Traf1 is essential for endogenous JNK activation and Drosophila development, whereas Traf2 is required for NF-kappaB signaling and activation of the antimicrobial immune system. Interestingly, Traf1 and Traf2 do not interfere in each other's signaling and consequent physiological activities. Therefore, it is concluded that Traf1 and Traf2 have independent roles in Drosophila by selectively regulating different downstream signaling pathways (Cha, 2003).

To understand the physiological roles of Traf1 and Traf2, these genes were overexpressed by using tissue-specific GAL4 drivers. While searching through the Berkeley Drosophila Genome Project P-element database, two P-element insertion lines, EP(2)0578 and EP(X)1516, were found with insertions at the 5' upstream region of the Traf1 and Traf2 gene, respectively. It was presumed that the insertion sites and directions of the EP elements in both EP fly lines are optimal for inducing Traf1 and Traf2 expression using tissue-specific GAL4 drivers (Cha, 2003).

In order to determine whether these EP lines indeed are capable of inducing the transcription of Traf1 and Traf2 genes, these lines were crossed with a hs-GAL4 line, and the mRNA levels for both DTRAFs after a heat shock were examined by Northern blot analysis. As expected, the Traf1 and Traf2 transcript levels were strongly increased by GAL4 inductions. These results confirm that the EP(2)0578 and EP(X)1516 lines are appropriate for overexpressing Traf1 and Traf2, respectively, at a specific location and time using various GAL4 drivers (Cha, 2003).

To investigate the consequences of ectopic expression of Traf1 in the developing Drosophila eye, Traf1 was overexpressed by using an eye-specific gmr-GAL4 driver. The eyes of adults carrying one copy each of both gmr-GAL4 and Traf1 show a rough-eye surface with disorganized arrays of ommatidia, whereas the eyes of flies carrying either one copy of gmr-GAL4 or one copy of Traf1 alone appear normal. Examination of the retinal sections of adults carrying both gmr-GAL4 and Traf1 reveals the number of ommatidia to be reduced and the number and shape of the photoreceptor cells in each ommatidium also to be abnormal, compared to the controls, which carry only the gmr-GAL4 driver (Cha, 2003).

When two copies of Traf1 were overexpressed in the eye, it displayed a more severe phenotype and a reduced number of ommatidia, resulting in a size reduction of the compound eye, and some ommatidia were fused with each other. In contrast, ectopically expressed Traf2 had no effect on the eye development; ommatidial array, bristles, and compound eye size were all found to be normal (Cha, 2003).

To determine which signaling pathway is activated and induces malformation of the optic system by ectopic expression of Traf1 in vivo, the genetic interactions were tested between mutants of various signaling pathways and a Traf1-overexpressing line (gmr>Traf1/+). Included in this screen were UAS lines that activate extracellular signal-regulated kinase (ERK), p38 MAP kinase, and JNK signaling pathway, respectively. Among the various overexpression lines tested, only Hemipterous (Hep; Drosophila homolog of MKK7 encoded by hemipterous [hep]) and Basket (Bsk; Drosophila homolog of JNK encoded by basket [bsk]) were found to interact genetically with Traf1. The UAS-hep or UAS-bsk itself under the control of gmr-GAL4 driver had no effect on eye morphogenesis. Coexpression of Bsk with Traf1 increased the disturbance of the ommatidial array in the compound eye in comparison to the eye phenotype resulting from one copy overexpression of Traf1. In addition, when Hep was coexpressed with Traf1, the number of ommatidia and the size of the compound eye were reduced more dramatically, which is very similar to the Traf1 two-copy expression phenotype. To further examine whether Traf1 signaling is mediated by Hep, Traf1 was expressed under a hemizygous hep mutant background. As a result, the abnormal ommatidial array of the compound eye was recovered to the level of the wild-type eye (Cha, 2003).

Interestingly however, Traf2, did not display any interaction with the ERK or the p38 MAP kinase pathway components, nor even with JNK pathway components. Tests were performed to see whether Traf2 exerts its effect on the eye development by interacting with Traf1. Coexpression of Traf2 with Traf1 in the Drosophila compound eye did not alter the rough-eye phenotype caused by a sole overexpression of Traf1. These results strongly support the view that Traf1 can activate the JNK signaling cascade in vivo and that Traf2 is not correlated with Traf1 signaling at all, at least during eye development (Cha, 2003).

Based upon the result that Traf1 is involved in JNK signaling, attempts were made to find out the signaling components between Traf1 and Hep by genetic interaction studies. Various kinases, such as Misshapen (msn), Slipper (slpr), and Drosophila Transforming growth factor ß-activated kinase 1 (Tak1), are known to be the upstream kinases for Hep in the eye development. Among these kinases, Tak1 synergistically increased the roughness of the compound eye surface and also reduced the eye size when coexpressed with Traf1. Moreover, Tak1-null mutation (Tak1¹), which has no effect on the eye morphology, is able to block the rough-eye phenotype caused by Traf1 overexpression. These data suggest that Traf1 activates the JNK signaling pathway via Tak1 and Hep (Cha, 2003).

In order to further confirm that Traf1 activates the JNK kinase signaling pathway at a molecular level, JNK activity in the eye discs was examined by two different experimental approaches -- an immunohistochemical assay with anti-phospho-specific JNK antibody and a puckered-LacZ reporter assay. JNK phosphorylation is highly induced by overexpression of Traf1 compared to the control. In addition, expression of puckered (puc), a well-known downstream target of JNK, is also highly induced by Traf1 compared to the control. Collectively, the results clearly demonstrated that Traf1 activates the JNK signaling pathway in vivo (Cha, 2003).

Apoptosis can be induced by the activation of the JNK pathway. Because ectopic coexpression of Traf1 with Hep has a synergistic effect on the reduction of ommatidia number and eye size, the rough-eye phenotype induced by Traf1 overexpression seemed to be a result of the Hep/JNK signaling-dependent apoptotic cell death. Therefore, whether overexpression of Traf1 can induce apoptosis in the eye disc cells was investigated by using TUNEL assay. In the discs of wild-type third-instar larvae, there are few apoptotic cells. In contrast, the eye imaginal discs from transgenic flies overexpressing Traf1 reveal a highly increased number of apoptotic cells in the region posterior to the morphogenetic furrow in a gene dosage-dependent manner. However, ectopic expression of Traf2 failed to induce apoptotic cell death, which is consistent with its inability to activate the JNK pathway (Cha, 2003).

Since hemizygous hep mutation (hep¹/Y) is sufficient to suppress the rough-eye phenotype caused by Traf1 overexpression, whether hep mutation can inhibit the Traf1-induced apoptotic cell death in the eye imaginal discs was examined. When TUNEL assay was performed against the eye discs of hep¹/Y; gmr>Traf1/± larvae, the number of apoptotic cells was dramatically decreased to almost wild-type levels. These results prove that Traf1 overexpression can activate the Hep/JNK signaling pathway and consequently induce apoptosis (Cha, 2003). Traf1^ex1, a loss-of-function allele for Traf1 gene, was generated through imprecise excision of the P-element in EP(2)0578 fly. RT-PCR analysis clearly demonstrate that the homozygous Traf1^ex1 mutant fails to produce Traf1 mRNA, indicating that Traf1^ex1 is a null allele for Traf1. The endogenous puckered transcription level was examined in the mutant larvae by RT-PCR analysis; the amount of puckered gene transcript was severely decreased in Traf1^ex1 mutant in comparison to wild-type larvae. This strongly implies the reduced JNK activity in Traf1^ex1 mutant (Cha, 2003).

In order to confirm this, the genetic interaction between the Traf1-null fly and a transgenic fly for JNK (ap>JNK^DN) was examined by observing the thorax closure phenotype. Thorax closure, the joining of the parts of the two wing imaginal discs during metamorphosis, is tightly controlled by the Drosophila JNK signaling pathway. When the activity of JNK pathway is downregulated by expressing a dominant-negative form of JNK on the thorax in ap>JNK^DN flies, the joining process is impaired and a cleft is formed at the dorsal midline in a gene dosage-dependent manner. Strikingly, a reduction of Traf1 gene dosage in heterozygous Traf1^ex1 dramatically enhances the thorax closure defect in ap>JNK^DN flies by expanding the cleft and also disrupting its notum structure, suggesting that Traf1 mutation leads to a greater reduction of the endogenous JNK activity in ap>JNK^DN flies. These data strongly support the critical roles of Traf1 in Drosophila development by positively modulating JNK signaling activities (Cha, 2003).

It is concluded that Traf1 can specifically activate the JNK signaling cascade. This conclusion is based on four lines of evidence: (1) ectopic expression of Bsk, Hep, or Tak1 with Traf1 exerts a highly synergistic effect on the rough-eye phenotype of Traf1 overexpressing flies; (2) disruption of hep or Tak1 function sufficiently suppresses the Traf1-induced eye defects; (3) histochemical analysis with either an anti-phospho-specific JNK antibody or the puckered-LacZ reporter system provides direct molecular evidence that Traf1 can induce phosphorylation and consequent activation of JNK; (4) Traf1 deficiencies in Traf1^ex1 mutants generate the same phenotypes detected in the loss-of-function mutants of the JNK signaling pathway, such as increased thorax closure defects and reduced puckered transcription (Cha, 2003).

In addition to its role in the JNK signaling pathway, previous results from cell culture experiments have implicated the involvement of TRAF in other MAP kinase signaling pathways. However, no evidence was found for the involvement of Traf1 in the p38- or the ERK-MAP kinase pathway in Drosophila. Specifically, overexpression of Traf1 with D-p38b or rl^Sem had no effect on the Traf1-induced eye phenotype. Likewise, ectopic expression of Traf1 with a dominant-negative form of D-p38b, D-p38b^DN, where Thr183 of the MAP kinase kinase target site is replaced with Ala, or the expression of Traf1 in a heterozygotic rl¹ genetic background also had no effect on the Traf1-induced rough-eye phenotypes. Taken together, these data strongly suggest that Traf1 specifically activates JNK signaling, but not the other MAP kinases (ERK- and p38-MAP kinase) signaling pathways, in Drosophila (Cha, 2003).

Ectopic Traf1 expression in imaginal eye discs induces apoptosis. Is the Traf1-induced apoptosis mediated through the JNK pathway? There are two observations strongly supporting this possibility: (1) phosphorylation of JNK and an increase in puckered gene expression were detected at the posterior region of eye discs, in which programmed cell death occurs most intensely by Traf1 expression; (2) the apoptosis induced by Traf1 overexpression is strongly suppressed by a loss-of-function allele of hep (Cha, 2003).

There are several supporting studies suggesting an involvement of the TRAF/JNK pathway in apoptosis in vertebrate cells. In addition, the Drosophila JNK pathway has been implicated in regulating apoptosis upon deregulation of Decapentaplegic (dpp), Wingless (wg), and integrin/tensin (int/ten) signalings in the developing wing discs. Moreover, it has been reported that overexpression of a constitutively activated form of Hep (hep^Act) or Jun (jun^Asp) can lead to eye phenotypes highly similar to Traf1 overexpression. Collectively, it is highly likely that TRAF regulates apoptosis through the JNK signaling pathway not only in vertebrates but also in flies (Cha, 2003 and references therein).

Interestingly, in mammals, TRAFs are thought to be involved in both JNK and NF-kappaB signalings to regulate various cellular responses. However, in Drosophila, Traf1 and Traf2 are involved in the regulation of separate signaling pathways and correspondingly serve different physiological functions. It is hypothesized that this difference between mammalian and Drosophila TRAFs is originated from structural differences of the N-terminal domains of TRAFs (Cha, 2003).

Cell culture-based studies have suggested that the structural differences among various TRAFs mainly exist in their RING finger and zinc finger domains, and these domains of the proteins must play essential roles in determining their downstream signaling pathways. In mammals, an intact RING finger domain has been shown to be required for the TRAF-mediated NF-kappaB activation but is dispensable for JNK signaling (Dadgostar, 1998). There was another report that zinc finger domains are responsible for membrane localization of TRAF and activation of the JNK pathway (Dadgostar, 2000). A forced localization of TRAF3 (which is normally unable to activate the JNK pathway) to the cell membrane by substitution of zinc finger domains was sufficient to convert this molecule into an activator of JNK (Cha, 2003).

Intriguingly, the two Drosophila TRAFs are distinguished from one another by the presence of a complete zinc finger domain or RING finger domain. Therefore, it is quite possible that the absence of the RING finger domain in Traf1 prevents the protein from interacting with the NF-kappaB pathway. However, Traf2 has fewer zinc finger domains than Traf1 or mammalian TRAFs, and this may inhibit Traf2 from its membrane localization and/or consequent activation of the JNK pathway. Further studies at a molecular level with various domain-modified DTRAF proteins should resolve this proposition (Cha, 2003).

GENE STRUCTURE

cDNA clone length - 2360 bp (transcript P1)

Bases in 5' UTR - 262

Exons - 2

Bases in 3' UTR - 537

PROTEIN STRUCTURE

Amino Acids - 486 (from transcript P1) and 412 (from transcript P2)

Structural Domains

A Drosophila gene encoding a protein homologous to the mammalian TRAF family was identified by searching the public data bases. The full-length Drosophila TRAF (Traf1) cDNA was isolated from a cDNA library prepared from 0-4-h Drosophila embryos. This Traf1 cDNA was contained within a unique exon located in the Drosophila chromosome 2L (24F1-24F2) and encoded a predicted protein of 387 amino acids in length. The AUG that initiates this open reading frame is present within a favorable context for translation. Traf1 contains seven zinc finger domains in its N-terminal region, followed by a TRAF domain located near the C terminus of the molecule. Thus, the domain topology of Traf1 is similar to other previously identified mammalian TRAF proteins. In contrast to many TRAFs, however, Traf1 does not contain a RING finger domain, and no evidence of a coiled-coil region upstream of the TRAF domain was found. Traf1 was also isolated based on its ability to interact with the Ste20 kinase Msn in a yeast two-hybrid screen (Liu, 1999). This Drosophila TRAF appears to be the same protein as reported in the Zapata (2000) study, except that it contains an additional 99 amino acids located at the N terminus of the protein, suggesting that the two proteins arise from a common gene (Zapata, 2000).

Using computer data base searches, TRAF proteins have also been identified in C. elegans and in Arabidopsis. The C. elegans TRAF contains a RING finger domain and five zinc fingers domains at the N terminus of the protein in addition to the TRAF domain at the C terminus. The Arabidopsis TRAF protein is an example of a family of hypothetical proteins containing TRAF domains found in Arabidopsis thaliana. Contrary to the other known TRAF proteins, the TRAF domain is located in the N terminus of this protein. The Arabidopsis TRAF protein does not contain either RING or zinc finger domains. The predicted amino acid sequence of Drosophila Traf1 is most similar to TRAF4 (47% identity) among the TRAFs, both when considering only the homology within the TRAF domain and when evaluating the entire molecule. The predicted amino acid sequence C. elegans TRAF shares similar extents of similarity with TRAF3, TRAF4, and TRAF5 (36% to 37% identity). Arabidopsis TRAF is most similar to TRAF3 (25% identity) (Zapata, 2000).

The sequences corresponding to the TRAF domains of Drosophila Traf1, C. elegans TRAF, Arabidopsis TRAF, and Dictyostelium TRAF were threaded onto the recently published x-ray crystal structure of human TRAF2. All of these TRAFs from lower organisms adopted the same overall fold as the TRAF domain of human TRAF2 (Zapata, 2000).

TNF-receptor associated factors (TRAFs) comprise a family of adaptor proteins that act as downstream signal transducers of the TNF receptor superfamily and the Toll/interleukin-1 receptor family. The mammalian TRAFs 2, 5 and 6 are known to activate JNK- and NF-kappaB signaling pathways, whereas the function of the other three mammalian family members, TRAF 1, 3 and 4 is less well characterized. Vertebrate TRAFs have a very similar structure with the exception of TRAF1: aside from the characteristic C-terminal TRAF domain, they share a N-terminal RING finger followed by five or, in the case of TRAF4, seven regularly spaced zinc fingers. Two TRAF homologs are present in the genome of Drosophila melanogaster: Traf1 and Traf2 (also known as Traf6) and both have been implicated in the Toll-receptor pathways leading to the activation of NF-kappaB and JNK. Traf1 is most closely related to mammalian TRAF4, which is predominantly expressed during nervous system development and in ephitelial progenitor cells. In order to gain insight into possible roles of Traf1 during development, a detailed transcriptional analysis of the gene was performed at various embryonic and larval stages (Preiss, 2001).

The Traf1 gene encodes two proteins of 402 and 487 amino acids that differ in their very N-terminus by the use of alternative first exons. The respective C-terminus (397 amino acids) is identical and comprises seven zinc fingers and the TRAF domain. Despite the lack of the amino-terminal ring finger, the structure of Traf1 resembles most closely that of mammalian TRAF4s. In fact, the identity between the complete amino acid sequences of Traf1 and human TRAF4 is the highest found between all known TRAF family members. Identity of 45% over the entire length and of 50% identity within the C-TRAF domain is observed. Thus, Traf1 is most likely the Drosophila homolog to vertebrate TRAF4 rather than to TRAF1. The second Drosophila TRAF (Traf2) is quite diverged: it contains a ring finger and two zinc finger motifs and has identity of up to 19% to mammalian TRAFs. In accordance with its role in the transduction of signals from Toll-receptors in the context of innate immunity in Drosophila (Kopp, 1999), Traf2 shows a rather low level, uniform expression throughout development. Mammalian TRAF4 is involved in signaling by the low affinity NGF receptor and is expressed in the developing central and peripheral nervous system in the mouse as well as in ephitelial progenitor cells. Therefore, TRAF4 is unique in its expression during nervous system development compared to other TRAFs (Preiss, 2001).

Both Traf1 and Traf2 have a TRAF domain at the C terminus, but Traf1 has seven repeated zinc finger domains at the N terminus, in contrast to Traf2, which has a single RING finger domain, as well as two zinc finger domains (Cha, 2003).

date revised: 28 December 2003

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