Traf1 interacts with Pelle and regulates NFkappaB activity

In the following analyzis Zapata, 2000, demonstrates that Traf1 interacts with Pelle and regulates NFkappaB activity. Chi, 2003, maintains that this is a function of Traf2, and that Traf1 function is restricted to the JNK pathway. Zapata's analysis is presented below to allow comparison with Chi's analysis as presented in the Biological Overview. Chi's evidence that Traf1 does not induce NF-kappaB activation and the consequent in vivo NF-kappaB-dependent immune responses is presented in the Evolutionary Homologs section.

A member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been identified in Drosophila. Traf1 contains 7 zinc finger domains followed by a TRAF domain, similar to mammalian TRAFs and other members of the family identified in data bases from Caenorhabditis elegans, Arabidopsis, and Dictyostelium. Analysis of Traf1 binding to different members of the human TNF receptor family has shown that this protein can interact through its TRAF domain with the p75 neurotrophin receptor and weakly with the lymphotoxin-ß receptor. Traf1 can also self-associate and binds to human TRAF1, TRAF2, and TRAF4. Interestingly, Traf1 interacts with human cIAP-1 and cIAP-2 but not with Drosophila DIAP-1 and -2. By itself, Traf1 does not induce significant NFkappaB activation when overexpressed in mammalian cells, although it specifically increases NFkappaB induction by TRAF6. In contrast, TRAF2-mediated NFkappaB induction is partially inhibited by Traf1. Mutants of Traf1 lacking the N-terminal region inhibit NFkappaB induction by either TRAF2 or TRAF6. Traf1 specifically associates with the regulatory N-terminal domain of Pelle, a Drosophila homolog of the human kinase interleukin-1 receptor-associated kinase (IRAK). Interestingly, though Pelle and Traf1 individually are unable to induce NFkappaB in a human cell line, co-expression of Pelle and Traf1 result in significant NFkappaB activity. Interactions of Traf1 with human TRAF-, TNF receptor-, and IAP-family proteins imply strong evolutionary conservation of TRAF protein structure and function throughout Metazoan evolution (Zapata, 2000).

To investigate whether Traf1 can interact with the cytosolic domains of various members of the TNFR family, GST fusion proteins containing the cytoplasmic domains of CD40, p75NGFR, LTßR, and DR4 were tested for their ability to bind an in vitro translated 35S-labeled Traf1 (Delta1-124) fragment that includes the TRAF domain. Traf1 (Delta1-124) specifically binds to the cytosolic tail (ct) of p75NGFR and more weakly to LTßR(ct). This pattern of binding is similar to that of TRAF4, which also binds specifically to these two members of the TNFR family in vitro (Krajewska, 1998). This observation is consistent with the more extensive sequence similarity of Traf1 with TRAF4 (Zapata, 2000).

The ability of the GST-Traf1 (Delta1-124) fusion protein to interact with the six known human TRAFs and to itself was examined. Traf1 binds to TRAF1, TRAF2, and TRAF4, whereas no binding was detected to TRAF3, TRAF5, or TRAF6. Also, DTRAF is able to self-associate, similar to other TRAF family proteins (Zapata, 2000).

Another type of protein that has been shown to interact with the TRAF domain of TRAF2 is IAP-family proteins, specifically cIAP-1 and cIAP-2. IAP family proteins are involved in regulating cell death by direct inhibition of caspases. Two members of this family have been identified in Drosophila. An examination was made of the ability of the TRAF domain of Traf1 to interact with all known human members of the IAP family as well as baculovirus CpIAP and the two Drosophila IAPs, DIAP-1 and DIAP-2. Traf1 (Delta1-124) interacts with human cIAP-1 and weakly with cIAP-2. In contrast, none of the other IAPs tested, including DIAP-1 and DIAP-2, binds to Traf1. Thus, Traf1 may not be a regulator of IAPs in Drosophila, although it evidently shares structural features in common with TRAF2 that permit binding to cIAP-1 and cIAP-2 (Zapata, 2000).

Mammalian TRAF2, TRAF5, and TRAF6 induce NF-kB activation when overexpressed in cells. To investigate if Traf1 has a role in regulating NFkappaB activity, Traf1 and the deletion mutants Traf1 (Delta1-124) and Traf1 (Delta1-226) were overexpressed in human epithelial 293T cells, together with a NFkappaB-responsive reporter plasmid. None of these proteins induced significant NFkappaB activity in this cell line. However, when Traf1 was co-transfected with TRAF6, a clear increase in NFkappaB activity was detected as compared with the levels of NFkappaB activity generated by TRAF6 alone. In contrast, co-transfection of TRAF6 with the Traf1 deletion mutants dramatically reduces TRAF6-mediated NFkappaB. Interestingly, co-expression of TRAF2 with either full-length Traf1 or the deletion mutants results in an inhibition of the TRAF2-mediated NFkappaB induction. The levels of expression of these proteins in 293T cells after transfection were examined by immunoblotting, revealing that DTRAF(Delta1-124) does not alter the levels of TRAF2 or TRAF6. Since Traf1 binds TRAF2 but not TRAF6 in vitro, these data suggest that full-length Traf1 may interfere with TRAF2 function through direct interaction. In contrast, the effects of Traf1 on TRAF6 are presumably indirect, perhaps reflecting an interaction of Traf1 with signal transducing proteins downstream of TRAF6 (Zapata, 2000).

Although TRAF2, TRAF5, and TRAF6 have been shown to activate NFkappaB via the kinases NFkappaB-inducing kinase and receptor interacting protein, TRAF6 has also been shown to regulate NFkappaB through the kinase IRAK (Cao, 1996). IRAK is the human counterpart of the Drosophila kinase Pelle and mediates signal transduction by the Toll/interleukin 1 receptor family. In Drosophila, the transmembrane receptor signaling by Toll is mediated by two proteins, Tube and Pelle, the latter representing the Drosophila counterpart of mammalian IRAK kinase. To determine if the TRAF domain of Traf1 can interact with components of the Drosophila Toll signal transduction pathway, yeast two-hybrid assays were performed. These two-hybrid experiments revealed a strong interaction of Traf1 (Delta1-124) with Pelle but not with Tube or Toll. The interaction of Traf1 (Delta1-124) with Pelle is mediated by the N-terminal regulatory domain of Pelle, which contains a death Domain, and not by the C-terminal protein kinase domain, similar to the interaction previously described for Pelle and Tube. The interaction of Traf1 with Pelle was further demonstrated by in vitro binding assays. In vitro translated Pelle specifically binds to a GST-Traf1 (Delta1-124) fusion protein, whereas Tube does not (Zapata, 2000).

Next, whether Traf1 can regulate NFkappaB induction by Pelle was examined. When expressed in HEK293T cells, neither Pelle nor any of the Traf1 constructs induce an increase in NFkappaB activity. However, when Pelle and Traf1 are co-transfected, a clear increase in NFkappaB activity is detected. This induction of NFkappaB is not observed when Pelle is co-transfected with Traf1 (Delta1-124) or Traf1 (Delta1-236), suggesting an important role for the zinc finger domains of Traf1 in this co-stimulatory effect. Moreover, both Traf1 (Delta1-124) and Traf1 (Delta1-236) are able to abrogate the induction of NFkappaB activity mediated by co-transfection of Pelle and Traf1, suggesting that these two Traf1 deletion mutants function in a dominant negative fashion. The induction of NFkappaB activity in 293T cells by the combination of Traf1 and Pelle is specific in that reporter genes containing other promoters (e.g., p53-responsive; retinoid-responsive; androgen-responsive) are not affected (Zapata, 2000).

Altogether these results demonstrate that Traf1 is able to regulate NFkappaB activation in collaboration with Pelle, suggesting that Traf1 is a component of the Toll pathway in Drosophila. This pathway has been implicated in the regulation of the dorsal-ventral polarization of developing embryos. However, microinjection of Traf1 (1-124) mRNA into Drosophila embryos fails to affect the normal dorsal-ventral patterning, suggesting that Traf1 may not be required for this developmental process. This observation, together with the recent characterization of another member of the TRAF family in Drosophila implies that flies may contain other TRAF-family genes that create redundancy in the pathways available for NFkappaB induction, similar to the situation with mammalian TRAFs. A role for Traf1 in innate immune responses to pathogens in flies, however, remains to be explored (Zapata, 2000).

Traf1 binds the Ste20 kinase Misshapen and activates Jun kinase

Two families of protein kinases that are closely related to Ste20 in their kinase domain have been identified: the p21-activated protein kinase (Pak) and SPS1 families. In contrast to Pak family members, SPS1 family members do not bind and are not activated by GTP-bound p21Rac and Cdc42. A member of the SPS1 family, called Misshapen (Msn), has been placed genetically upstream of the c-Jun amino-terminal (JNK) mitogen-activated protein (MAP) kinase module in Drosophila. The failure to activate JNK in Drosophila leads to embryonic lethality due to the failure of these embryos to stimulate dorsal closure. Msn probably functions as a MAP kinase kinase kinase kinase in Drosophila, activating the JNK pathway via an as yet undefined MAP kinase kinase kinase. A Drosophila TNF-receptor-associated factor, Traf1, has been identified by screening for Msn-interacting proteins using the yeast two-hybrid system. In contrast to the mammalian TRAFs that have been shown to activate JNK, Traf1 lacks an amino-terminal 'Ring-finger' domain, and overexpression of a truncated Traf1, consisting of only its TRAF domain, activates JNK. Another DTRAF, Traf2, has been identified that contains an amino-terminal Ring-finger domain. Msn specifically binds the TRAF domain of Traf1 but not that of Traf2. The region between the kinase and C-terminal regulatory domains of Msn is sufficient to bind Traf1, whereas neither the kinase domain nor the C-terminal domain alone can bind the protein. A stretch of about 250 amino acids that lies in the N-terminal portion of the interdomain is sufficient for Msn to bind Traf1. The C-terminal portion of this region does not interact with Traf1 but does interact with the SH3 domains of Dreadlocks, supporting the idea that the central region couples Msn and related Ste20 kinases to multiple upstream targets. Traf1 and Traf2 can dimerize with themselves but cannot form heterodimers. In Drosophila, Traf1 is thus a good candidate for an upstream molecule that regulates the JNK pathway by interacting with, and activating, Msn. Consistent with this idea, expression of a dominant-negative Msn mutant protein blocks the activation of JNK by Traf1. Furthermore, coexpression of Msn with Traf1 leads to the synergistic activation of JNK. A truncated Msn lacking the kinase domain functions as a dominant-negative inhibitor by blocking activation of JNK by Traf1. Some of these observations are extended to the mammalian homolog of Msn, Nck-interacting kinase (NIK), suggesting that TRAFs also play a critical role in regulating Ste20 kinases in mammals (Liu, 1999).

Reaper-mediated inhibition of DIAP1-induced Traf1 degradation results in activation of Basket/JNK in Drosophila

Although Jun amino-terminal kinase (JNK) is known to mediate a physiological stress signal that leads to cell death, the exact role of the JNK pathway in the mechanisms underlying intrinsic cell death remains largely unknown. Through a genetic screen, it has been shown that a mutant of Drosophila tumor-necrosis factor receptor-associated factor 1 (Traf1) is a dominant suppressor of Reaper-induced cell death. Reaper modulates the JNK pathway through Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), which negatively regulates Traf1 by proteasome-mediated degradation. Reduction of JNK signals rescues the Reaper-induced small eye phenotype, and overexpression of Traf1 activates the Drosophila ASK1 (apoptosis signal-regulating kinase 1; a mitogen-activated protein kinase kinase kinase) and JNK pathway, thereby inducing cell death. Overexpresson of DIAP1 facilitates degradation of Traf1 in a ubiquitin-dependent manner and simultaneously inhibits activation of JNK. Expression of Reaper leads to a loss of DIAP1 inhibition of Traf1-mediated JNK activation in Drosophila cells. Taken together, these results indicate that DIAP1 may modulate cell death by regulating JNK activation through a ubiquitin-proteasome pathway (Kuranaga, 2002).

Three Drosophila genes, reaper, hid, and grim, have been identified as key regulators of apoptosis during Drosophila embryogenesis. Products of all three genes induce apoptosis through a pathway that requires activation of caspase. Through interactions mediated by the N terminus, each of these proteins binds to DIAP1. Genetic and biochemical data indicate that one way in which these proteins promote apoptosis is by inhibiting the ability of DIAP1 to prevent death-inducing caspase activity. Smac (also known as DIABLO) and HtrA2 (also known as Omi), mammalian mitochondrial proteins whose truncated N termini share similarity with Rpr, Hid and Grim, also inhibit the antiapoptotic function of XIAP and enhance caspase activation (Kuranaga, 2002).

To study the genetic regulation of this conserved cell death mechanism, a dominant modifier screen of Rpr was carried out that covered more than 70% of the Drosophila genome. Overexpression of Rpr using an eye-specific promoter (GMR) gave rise to dose-dependent cell death through caspase activation, resulting in flies with small eyes. Df(2L)sc19-8 was identified as a suppressor of Rpr through deletions covering the region 24C2-25C8. Because this suppressor line had a large deletion, lines with smaller deletions around the 24C2-25C8 region were subsequently screened, including Df(2L)ed-dp, Df(2L)dp-h25, Df(2L)M24F-B, Df(2L)tkv3 and Df(2L)ed1. Of all the strains examined, three overlapping deletions, Df(2L)ed-dp, Df(2L)dp-h25 and Df(2L)M24F-B, suppressed the Rpr-induced small eye phenotype: this information allowed a narrowing down of the suppressor region to 24E4-25A2. The ability of mutants covering the 24E4-25A2 region to improve the Rpr-induced small eye phenotype was tested: a P-element insertion, EP(2)578, in the first exon in the noncoding region of the Drosophila homolog of TRAF1 (Traf1) substantially suppresses the reduced eye size caused by Rpr (Kuranaga, 2002).

To assess the reduced expression of the Traf1 transcript in this homozygous mutant, polymerase chain reaction was carried out with reverse transcription (RT-PCR) using specific primers that detected Traf1 messenger RNA from wild-type and mutant third-instar larvae. The expression of Traf1 was markedly reduced in the homozygous mutant. Thus, Traf1 mutant was identified as a putative suppressor allele on Rpr (Kuranaga, 2002).

Df(3R)H-B79 was also identified using deletions covering the 92B3-F13 region. A search of the translated nucleotide databases, using the TBLASTN program, identified an expressed sequence tag (EST) clone, LD40486, that maps to this region (Berkeley Drosophila Genome Project) and has similarity to the kinase domain of the mitogen-activated protein kinasae kinase kinase (MAPKKK) family of serine/threonine kinases. Previously, the gene from the genomic region that encoded the MAPKKK had been isolated as PK92B, from an eye-antennal imaginal disc complementary DNA library using a PCR-based approach. It has an open reading frame (ORF) of 650 amino acids. The EST clone (LD40486) encoding the PK92B cDNA was analyzed and it can potentially encode a longer form of the protein (1,367 amino acids). Notably, the longest ORF of the cDNA showed substantial overall homology to the human MAPKKK ASK1, with the kinase domain of this cDNA showing 73% identity and 83% similarity with the amino acid sequences of ASK1 (Kuranaga, 2002).

To determine whether PK92B has a role in JNK activation in Drosophila, Basket/JNK and PK92B were coexpressed in Drosophila S2 cells along with the kinase-dead K618M mutant of PK92B. JNK activation by PK92B was suppressed by PK92BK618M in a dose-dependent manner, which suggests that PK92BK618M is a dominant-negative form of PK92B and that PK92B is involved in JNK signalling. The kinase-dead PK92BK618M mutant was used to test whether the kinase activity of PK92B is important in mediating Rpr-induced cell death; the Rpr-induced reduced eye size is visibly suppressed by coexpression of PK92BK618M (Kuranga, 2002).

To test whether the Drosophila Basket/JNK pathway is involved in Rpr-induced cell death, the genetic interactions of GMR-Rpr with several mutants or transgenic lines of the JNK signalling pathway were examined. Two mutants in the JNK pathway were tested -- basket2 (bsk encodes JNK) and hemipterous1 (hep1, hep encodes DJNK kinase) -- as well as two transgenic lines, UAS-DJNK DN (expressing a dominant-negative form of Basket) and UAS-Dp38 DN (expressing a dominant-negative form of Drosophila p38; Dp38). In flies with reduced JNK signals, but not in those with reduced p38 signals, the reduced eye size of the GMR-Rpr flies is visibly improved. The transient expression of Rpr strongly activates Basket in third-instar larvae. These results suggest that Rpr can activate the JNK pathway, probably through Traf1 and PK92B (Kuranga, 2002).

In mammals, ASK1 interacts with members of the TRAF family and is sufficient and necessary for the activation of JNK induced by TNF-alpha and TRAF2. Traf1 has been reported to be involved in JNK activation. Therefore it was asked whether Traf1 and PK92B could activate DJNK in Drosophila S2 cells. These cells were transfected with PK92B or Traf1, and phosphorylation of Basket was detected by immunoblotting using an antibody against phosphorylated Basket. Both PK92B and Traf1 strongly induce Basket phosphorylation. Whether Traf1 could activate PK92B was examined. Traf1 and Flag-PK92B were cotransfected into S2 cells, and Flag-PK92B was immunoprecipitated with an antibody against Flag. The activation status of PK92B was then assessed using immunoblotting analysis with an antibody specific for phosphorylated ASK1. Consistent with the ability of Traf1 to activate Basket, PK92B is strongly activated by the coexpression of Traf1. A Basket-PK92B interaction was observed in S2 cells. Thus, it is possible that PK92B is a downstream target of Traf1 and that Basket is activated through a direct interaction with PK92B (Kuranga, 2002).

How Rpr affects the Traf1/PK92B/Basket signalling pathway was investigated. Because DIAP1 is a molecular target of Rpr, whether DIAP1 might affect the DJNK activation pathway was investigated. Traf1 was transfected into S2 cells with or without DIAP1 and the viability of cells was examined. Traf1-induced cell death was markedly suppressed by DIAP1. To confirm that Traf1-induced cell death is suppressed by DIAP1, a strain of UAS-Traf1 transgenic flies was generated. The overexpression of Traf1 driven by GMR-GAL4 causes a rough and small eye phenotype and increases the number of dying cells in the third-instar larval eye disc. The massive cell death caused by expression of Traf1 was mediated by the JNK pathway in the fly eye, because the Traf1-induced small eye phenotype is markedly improved in a heterozygous hep1 mutant background. The coexpression of DIAP1 substantially suppresses the Traf1-induced rough eye phenotype (Kuranga, 2002).

Next, the phosphorylation of Basket was examined by immunoblotting with an antibody against phosphorylated JNK in S2 cells. Activation of Basket by Traf1 is suppressed by DIAP1 in a dose-dependent manner, but DIAP1 does not affect the activation of Basket by PK92B. Notably, increased amounts of DIAP1 lower the amounts of Traf1. DIAP1 can directly interact with Traf1, which suggests that the downstream target of DIAP1 is Traf1, and not PK92B or Basket. DIAP1 and DIAP2, mammalian cIAP1 and cIAP2, and X-linked IAP (XIAP) all possess RING-finger and baculovirus IAP repeat (BIR) domains. The BIR domain is required for caspase binding and inhibition, whereas ubiquitination by several E3 ubiquitin ligases is dependent on the RING-finger motif. Notably, cIAP1 and XIAP catalyse their own ubiquitination in a manner dependent on their RING domain. It was therefore thought that the Traf1-DIAP1 interaction might represent ubiquitination of Traf1 by DIAP1. This possibility was tested by transfecting a fusion protein of Traf1 and hemagglutinin A (Traf1-HA), HA-ubiquitin and Flag-DIAP1 into S2 cells, and then carrying out immunoprecipitations and immunoblotting with an antibody against HA to examine the amount of ubiquitinated Traf1. Traf1 was heavily ubiquitinated in the presence of DIAP1 in a dose-dependent manner. Taken together, these results suggest that DIAP1 stimulates Traf1 degradation through ubiquitination. This regulation of Traf1 would therefore prevent Traf1-induced JNK activation as well as cell death (Kuranga, 2002).

Although Rpr could interact with DIAP1 through its N-terminal region, no direct interaction between Rpr and components of the JNK pathway (Traf1, PK92B and DJNK) was detected, which suggests that Rpr may be activating the JNK pathway through DIAP1. In agreement with this, expression of a Rpr mutant truncated at its N terminus (UAS-RprN) using the hs-GAL4 driver in third-instar larvae failed to activate DJNK, suggesting the importance of the Rpr and DIAP1 interaction for both caspase and DJNK activation. It has been shown that Rpr not only inhibits IAP function but also promotes the degradation of DIAP119-23. On the basis of these observations, it was reasoned that the degradation of DIAP1 by Rpr might be able to promote JNK activation. To assess this possibility, whether Rpr could prevent the degradation of Traf1 mediated by DIAP1 was examined. Not only did the expression of Rpr substantially inhibit the DIAP1-mediated degradation of Traf1, but it also activated Basket/JNK. These results suggest that expression of Rpr can stimulate activation of JNK by degrading DIAP1 and subsequently stabilizing Traf1 (Kuranga, 2002).

To determine the endogenous function of Traf1, the phenotype of adult flies that were homozygous for the Traf1 mutant, Traf1EP(2)578, was examined. The only marked characteristic of these flies was additional numbers of adult dorsal bristles. The external sensory organ on the notum is a typical structure of the Drosophila peripheral nervous system, where four large bristles (macrochaetes) are always observed in the wild-type scutellum. The ectopic bristles seen in Traf1EP(2)578 flies are probably the result of altered caspase activation, because they are also found in Drosophila Apaf-1 mutants, in flies that ectopically express caspase inhibitory proteins p35 and DIAP1, and in a mutant for a ubiquitin-conjugating enzyme that promotes the degradation of DIAP1. These results suggest that Traf1 and DIAP1 affect the activation of caspase in order to regulate the number of macrochaetes in adults; thus, Traf1 probably affects processes involving caspase-mediated events. The possibility that Traf1 has a role in the canonical JNK pathway cannot be ruled out, because the Traf1EP(2)578 mutant allele that was used in this study may not be a null allele (Kuranga, 2002).

In summary, it is proposed that Basket/JNK activity can be negatively regulated by DIAP1. Overexpression of DIAP1 prevents Traf1-induced Basket/JNK activation through degradation of Traf1. Rpr facilitates the degradation of DIAP1 through the ubiquitin-proteasome system, thereby inducing activation of Basket/JNK mediated by Traf1 and PK92B. These findings suggest that the degradation of IAPs in cells that have been instructed to undergo cell death may represent an evolutionarily conserved mechanism to facilitate cell death (Kuranga, 2002).

Localized JNK signaling regulates organ size during development

A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).

Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).

The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).

Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bskRNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bskDN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hepr75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bskDN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bskDN), total animal body size is not altered (Willsey, 2016).

To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).

In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).

A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).

To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with junRNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).

In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).

It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).

Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).

While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>CiACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).

To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).

dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).

Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).

Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).

TNF-receptor-associated factor 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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