Lic regulates JNK‐mediated cell death in Drosophila

Abstract Objectives The evolutionary conserved JNK pathway plays crucial role in cell death, yet factors that modulate this signalling have not been fully disclosed. In this study, we aim to identify additional factors that regulate JNK signalling in cell death, and characterize the underlying mechanisms. Materials and Methods Drosophila were raised on standard media, and cross was carried out at 25°C. The Gal4/UAS system was used to express proteins or RNAi in a specific temporal and spatial pattern. Gene expression was revealed by GFP fluorescence, X‐gal staining or immunostaining of 3rd instar larval eye and wing discs. Cell death was visualized by acridine orange (AO) staining. Images of fly eyes and wings were taken by OLYMPUS microscopes. Results We found that licorne (lic) encoding the Drosophila MKK3 is an essential regulator of JNK‐mediated cell death. Firstly, loss of lic suppressed ectopic Egr‐triggered JNK activation and cell death in eye and wing development. Secondary, lic is necessary for loss‐of‐cell polarity‐induced, physiological JNK‐dependent cell death in wing development. Thirdly, Lic overexpression is sufficient to initiate JNK‐mediated cell death in developing eyes and wings. Furthermore, ectopic Lic activates JNK signalling by promoting JNK phosphorylation. Finally, genetic epistatic analysis confirmed that Lic acts in parallel with Hep in the Egr‐JNK pathway. Conclusions This study not only identified Lic as a novel component of the JNK signalling, but also disclosed the crucial roles and mechanism of Lic in cell death.


| INTRODUC TI ON
Programmed cell death (PCD), or apoptosis, is a fundamental biological phenomenon in which cell death is genetically and biochemically regulated. Dysregulation of apoptosis will trigger a series of disorders that result in many types of diseases such as neurodegenerative diseases, immunodeficiency diseases and tumours. [1][2][3][4] Apoptosis is a strictly controlled process involving a series of gene activation, while expression and regulation of these genes are highly conserved among species, such as Bcl-2 family, Caspase family and oncogene such as c-myc 5 and tumour suppressor gene p53. 6 The JNK signalling is a very important pathway involved in the regulation of cell death, 7 migration, proliferation 8 and differentiation, 7 as well as cell morphology maintenance, cytoskeleton construction and other biological processes. 1,9 Drosophila is one of the best model organisms to study genetics, and many of the JNK pathway components and regulators have been identified from genetic screens in Drosophila. [10][11][12] The stress-activated protein kinase (SAPK) signalling consists of the JNK and p38 pathways. Its primary function is to respond to a series of environmental stresses (nutrients, osmotic pressure, temperature, etc) and participate in the regulation of apoptosis, proliferation, differentiation and other responses to adapt to changes in the external environment to ensure the proper function of the body. 13 In Drosophila, p38 is activated via dual phosphorylation at the Thr-Gly-Tyr motif by a specific MAPKK, Licorne (Lic), which encodes the fly ortholog of MKK3, 14 whereas the fly JNK, Bsk, is phosphorylated and activated by Hemipterous (Hep) encoding the MKK7 ortholog.
In 2009, Caroline Baril et al 15 found that mutant of Alphabet (Alph), a serine/threonine phosphatase belongs to the Drosophila protein phosphorylation 2C (PP2C) family, was able to rescue the lethality caused by hep or lic mutation. Genetic epistatic analysis confirmed that Alph acts as a negative regulator upstream of Hep and Lic in the MAPK pathway. In addition, both p38 and JNK signalling could be triggered by similar activators, such as pro-inflammatory cytokines (TNFα and IL-1) and stress stimulation (UV, H 2 O 2 and heat shock). 16 These results suggest that the two pathways may have overlapping or redundant functions, which might be achieved by sharing component(s). However, despite the reported role of Lic in p38 signalling, it remains elusive whether Lic also regulates JNK signalling in vivo.
We have previously carried out a genetic screen in Drosophila to search for additional regulators of Egr-triggered JNK-mediated cell death. 12,17 In this study, we provide genetic evidences demonstrating that lic encodes an essential component of the Egr-JNK pathway involved in cell death. We found that Lic is necessary for ectopic Egr-induced or loss-of-cell polarity-induced JNK activation and cell death. Ectopic Lic is sufficient to promote JNK phosphorylation, which activates JNK signalling and triggers cell death in development. Genetics data suggest Lic acts in parallel with Hep as a JNK kinase. However, it deserves further investigation whether MKK3 could phosphorylate and activate JNK signalling in mammal.

| Drosophila stocks and genetics
Stocks were raised on standard Drosophila media, and crosses were performed at 25°C. The following stocks were described previ- Haiyun Song and has been previously described. 29 lic mutant clones were generated with the MARCM system 30 and labelled by GFP expression (lic d13 , FRT19A/tub-Gal80, hs-Flp, FRT19A; act-Gal4, UAS-GFP/+). Flp recombinase was expressed conditionally using hs-Flp.
Heat shocks were performed at 37°C for 15 minutes during the first or second instar larval stage.

| Antibodies
The following primary antibodies were used for immunostaining:

| Image of fly eyes and wings
Three-day-old flies were collected and frozen at −80°C. When taking pictures, flies were unfrozen at room temperature and placed on 1% agarose plate. Light images of eye were taken by OLYMPUS stereo microscope SZX16 (Olympus Corporation, Shinjuku, Tokyo, Japan). Wings were dissected and placed on slide with alcohol/glycerol (1:1) buffer.
Light images of wing were taken by OLYMPUS BX51 microscope.

| Lic is essential for ectopic Egr-induced cell death in eye development
Egr is the fly ortholog of tumour necrosis factor (TNF) 32 and a wellrecognized upstream regulator of the JNK signalling pathway. 33 Egr is reported to play important roles in the regulation of cell proliferation, differentiation, apoptosis and immunity. 34 Compared with the control ( Figure 1A,G), ectopic expression of Egr in the developing eye driven by GMR-Gal4 (GMR>Egr) triggers JNK-mediated cell death in the eye imaginal disc ( Figure 1H,T) and produces a small eye phenotype in the adult ( Figure 1B,S). 35 We found that Egr-triggered small eye phenotype was effectively suppressed by knocking-down lic with two independent RNAi lines ( Figure 1D,E,S), but remained unaffected by expressing a GFP-RNAi control transgene ( Figure 1C,S).
Expression of the lic-RNAi by itself did not produce any discernible eye phenotype ( Figure S1a,b). lic d13 is a P element-induced allele of lic, with a deletion of 1411 nucleotides that removes the initiating methionine and the first 117 amino acids of the lic coding sequence. 29 Consistent with the RNAi data, GMR>Egr-induced small eye phenotype was effectively suppressed in heterozygous lic d13 mutants ( Figure S2b Oneway ANOVA test was used to calculate statistical significance, mean + SD, ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0. cell death in the eye disc, indicated by acridine orange (AO) staining, was markedly inhibited by mutation or RNAi-mediated depletion of lic ( Figure 1J,K,T and Figure S2d,f) and Bsk DN ( Figure 1L), but not that of GFP-RNAi ( Figure 1I). Together, these data suggest that Lic is necessary for ectopic Egr-induced cell death in Drosophila eye development.

| Lic is required for ectopic Egr-induced JNK pathway activation in developing eyes
Given that Lic is required for Egr-induced JNK-dependent cell death, we wonder whether Lic is necessary for Egr-induced JNK pathway activation. To this end, we examined the expression of puc-LacZ, a well-known reporter that characterizes the activity of JNK signalling. 35,36 Compared with control discs (Figure 1M), GMR>Egr induced strong puc-LacZ expression posterior to the morphogenetic furrow (MF) in eye discs ( Figure 1N), which was significantly suppressed by expressing two lic-RNAi ( Figure 1P,Q) or Bsk DN ( Figure 1R), but not the GFP-RNAi ( Figure 1O). Thus, Lic is necessary for ectopic Egr-induced JNK signalling activation in developing eyes.

| Lic is necessary for Egr-induced JNK-mediated cell death in wing development
To investigate whether Lic modulates Egr-induced JNK activation and cell death in other tissues, we turned to the wing imaginal discanother in vivo system that has been widely used to study signal transduction pathway. Ectopic expression of Egr driven by ptc-Gal4

| Lic is required for physiological JNK-mediated cell death
The above data suggest that Lic is necessary for ectopic Egr-induced JNK activation and cell death. Next, we wanted to verify whether Lic is involved in physiological activation of JNK signalling. Previous studies have found that disruption of cell polarity caused JNK-mediated cell death. 37,38 Under the control of the ptc promoter, RNAi-mediated downregulation of scrib generated the loss-of-ACV phenotype in adults ( Figure 3B,F), which is resulted from extensive cell death at the A/P boundary of the wing disc ( Figure 3H,L). We found that depletion-of-scrib-induced ACV loss and cell death were effectively inhibited by knocking-down lic or expressing Bsk DN that served as a positive control ( Figure 3C-F,I-L). Collectively, these data suggest that Lic is essential for physiological JNK-mediated cell death.

| Lic sufficiently activates JNK signalling
To investigate whether Lic is sufficient to activate JNK signalling, we overexpressed Lic in various regions of the wing disc. Ectopic expression of Lic along the A/P boundary driven by dpp-Gal4 ( Figure   S4a) or in the wing pouch by sd-Gal4 ( Figure S4d

| Lic regulates JNK phosphorylation
In Drosophila, JNK is known to be activated through phosphorylation by the MKK7 ortholog Hep. 39 While Lic encodes the Drosophila ortholog of MKK3 that has been implicated in the p38 signalling, 14,40 its role in JNK signalling has not been previously reported. We found that ptc>Egr-induced JNK phosphorylation ( Figure 4B Figure 5A,B), ectopic Lic was able to trigger JNK phosphorylation along the A/P boundary in the wing pouch ( Figure 5C,D), which was further confirmed by Western blot analysis ( Figure S5).
Intriguingly, the width of the GFP stripe was significantly reduced upon Lic expression, presumably due to Lic-induced cell death that was blocked by co-expression of p35 ( Figure S6d). Puc is a serine/ threonine protein phosphatase that blocks JNK activity through dephosphorylation. We found that expression of Puc dramatically suppressed ectopic Lic-induced JNK phosphorylation and restored the GFP stripe width ( Figure 5E,F). Collectively, these data suggest that Lic regulates Egr-induced JNK phosphorylation.

| Lic induces JNK-dependent cell death in development
Given that Lic can sufficiently activate JNK signalling, we wonder whether Lic is able to elicit JNK-dependent cell death. To this end, we expressed Lic along the A/P boundary in 3rd instar wing discs by ptc-Gal4. Previous work reported that activation of JNK by ptc>Hep resulted in cell death in wing discs and ACV loss in adult wings. 41 We found that, compared with the ptc>GFP control ( Figure 6A,G), ptc>Lic was able to trigger cell death in wing discs and produce the loss-of-ACV phenotype in adults ( Figure 6C,I). Both phenotypes could be further enhanced by a mutation in the endogenous puc, but blocked by the expression of Bsk DN ( Figure 6D-F,J-L). Intriguingly, expression of MKK3 was able to trigger JNK-dependent cell death in the wing disc ( Figure S7). Collectively, the data suggest that Lic is sufficient to induce JNK-dependent cell death in wing development, and this function is retained by MKK3.
To test whether Lic promotes JNK-mediated cell death in other tissues, we ectopically expressed Lic in the developing eye or scutellum by the ey-Gal4 or pnr-Gal4 driver. Compared with the controls, expression of Lic resulted in reduced organ sizes, which were significantly suppressed by expressing Bsk DN or Puc ( Figure   S8a,c-f and g, i-l), indicating that Lic triggers JNK-dependent cell death in a non-tissue-specific manner. Importantly, expressing a kinase-dead version of Lic (Lic KD ) 42 failed to trigger JNK phosphorylation ( Figure 4A) and cell death ( Figure 6H) or produce any discernible phenotypes ( Figure 6B and Figure S8b,h), suggesting the kinase activity is indispensable for Lic to induce JNK-mediated cell death.

| Lic acts in parallel with Hep to promote JNKmediated cell death
So far, our data suggest Lic is an essential component that acts downstream of Egr but upstream of Bsk in the Egr-JNK pathway.
As both Lic (Drosophila MKK3) and Hep (Drosophila MKK7) belong to the mitogen-activated protein kinase family (MAPKKs), and both are able to promote JNK phosphorylation, we assume Lic may act in parallel with Hep, and downstream of the MAPKKKs dTAK1 and Wnd. 23 To test this hypothesis, we performed genetic epistasis analysis between Lic and known kinases in the Egr-JNK pathway by using the loss-of-ACV phenotype, which had been the most constant and sensitive phenotype in our hands. As mentioned before, the ACV loss phenotype produced by ptc>Lic was significantly inhibited by expressing Bsk DN ( Figure 6C,E), indicating Lic acts upstream of Bsk. In addition, this phenotype was fully suppressed by expressing two independent lic-RNAi, but not GFP ( Figure 7A,B,I and Figure S9b), which served as the positive and negative controls, respectively. Consistent with the hypothesis, Lic-induced loss-of-ACV phenotype was not suppressed by depleting dTAK1, wnd, hep or mkk4, and vice versa, Hep-induced ACV loss could not be recovered by depleting lic (Figure 7C-E,G-I and Figure S9c). Furthermore, ectopic expression of Lic or Hep of Lic and Bsk failed to produce this phenotype ( Figure 7N).
Interestingly, loss of lic could impede ectopic dTAK1-but not Wnd-triggered small eye phenotype ( Figure S10), suggesting Lic mediates dTAK1-induced cell death. Based on the above evidences, we conclude that Lic most probably acts in parallel with Hep to promote JNK-mediated cell death.
As Lic has previously been reported as a MAPKK for the p38 kinase, we examined whether Lic activates JNK signalling through p38, or via a mechanism that is independent of p38. The Drosophila genome encodes three p38 family members, designated as p38a, p38b and p38c, 43 with p38b being proposed to play a central role in Drosophila p38 signalling. 44 We found that ptc>Lic-induced lossof-ACV phenotype was not affected by knocking-down p38a, p38b or p38c ( Figure 7F,I and Figure S9d-g), suggesting Lic regulates JNKmediated cell death in a p38-independent manner.

| Lic modulates physiological JNK activity
Endogenous JNK signalling is required for the thorax closure process in normal development, while impaired JNK activity results in a thorax cleft phenotype in the adults. 10

| DISCUSS IONS
lic encodes the Drosophila ortholog of MKK3, which has been previously reported as the MAPK kinase modulating p38 signalling in cell growth, stress response, innate immunity and asymmetric egg development in oogenesis. 46  Hep exhibited synergistic effect in promoting cell death ( Figure 7M).
The p38 mitogen-activated protein kinase (MAPK) pathway is activated in response to a variety of environmental stresses. 50 Although p38 signalling has been previously implicated in apoptosis, contradictory results suggest p38 could function as a positive or negative regulator. 51-53 JNK signalling plays a pro-apoptotic role in both mammalian and Drosophila systems. 54 Our study shows that ectopic expression of Lic promotes JNK phosphorylation and puc transcription, both are read-out of JNK signalling activation. Lic-induced apoptosis, which is independent of p38 but JNK-dependent, reminds us to pay more attention to the role of Lic in cell death and other JNK-related physiological process such as tumour development. Finally, it remains intriguing whether MKK3 participates in JNK signalling in mammals.

ACK N OWLED G EM ENTS
We thank Bloomington, VDRC, National Institute of Genetics (NIG-FLY) and Dr. Haiyun Song for fly stocks and reagents, and members of the Xue laboratory for comments and discussion. This work is supported by the National Natural Science Foundation of China (31571516, 31771595) and Shanghai Committee of Science and Technology (09DZ2260100, 18430711600 and 18140900400).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.