Abstract
Insect wing development is a fascinating and intricate process that involves the regulation of wing size through cell proliferation and apoptosis. In this study, we find that Ter94, an AAA-ATPase, is essential for proper wing size dependently on its ATPase activity. Loss of Ter94 enables the suppression of Hippo target genes. When Ter94 is depleted, it results in reduced wing size and increased apoptosis, which can be rescued by inhibiting the Hippo pathway. Biochemical experiments reveal that Ter94 reciprocally binds to Mer, a critical upstream component of the Hippo pathway, and disrupts its interaction with Ex and Kib. This disruption prevents the formation of the Ex-Mer-Kib complex, ultimately leading to the inactivation of the Hippo pathway and promoting proper wing development. Finally, we show that hVCP, the human homolog of Ter94, is able to substitute for Ter94 in modulating Drosophila wing size, underscoring their functional conservation. In conclusion, Ter94 plays a positive role in regulating wing size by interfering with the Ex-Mer-Kib complex, which results in the suppression of the Hippo pathway.
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Introduction
Wings are vital organs for insects, which participate in multiple processes, such as foraging and mating. Unlike the wings of birds, insect wings are usually formed through metamorphosis from the wing discs in larva1,2. This process involves cell proliferation and apoptosis, which are controlled by the Hippo pathway3,4. The core of the Hippo pathway consists of a series of kinases and the transcriptional cofactor Yorkie (Yki)5,6. The Ste20-like protein kinase Hippo (Hpo) complexes with Salvador (Sav) to phosphorylate and activate the downstream kinase Warts (Wts). Next, with assistance of the adaptor Mats, Warts directly binds and phosphorylates Yki on several serine residues, leading to its cytoplasmic retention7,8. When this kinase cascade is inactivated, unphosphorylated Yki enters the nucleus to turn on the expression of target genes9. As a matter of fact, due to the lack of DNA-binding domains, Yki only works synergistically with other transcription factors, most notably Sd10,11. In general, Yki enables the promotion of cell division and the suppression of cell death by activating the expression of pro-proliferative and anti-apoptotic genes12,13. In addition to inhibiting Yki’s entry into the nucleus, our recent studies also reveal that the Hippo pathway promotes Yki degradation through dissociating the Yki-Usp7 interaction14. Thus, the Hippo pathway governs Yki’s activity, at least through dual mechanisms, to regulate cell proliferation and death. In Drosophila, abnormal expression of any component of the core kinase cassette leads to wing defects, reflecting its important role for wing development15,16.
In Drosophila, there are two main branches upstream of the core kinase cassette: the Fat (Ft)-Dachsous (Ds) complex and the Ex (Expanded)-Mer (Merlin)-Kib (Kibra) complex5. Both Ft and Ds are transmembrane cadherins, that can independently activate the Hippo pathway, or work together to strengthen signaling through cell-cell contacts17. Ex, Mer and Kib form a ternary complex localized to the apical domain of epithelial cells to activate the Hpo-Wts kinase cascade18,19. Loss of Mer and Kib alone in Drosophila eyes fails to cause obvious defects, while their simultaneous loss leads to severe overgrowth, accompanied by activation of Hippo-responsive genes18. Another study has demonstrated that Mer and Ex work synergistically to activate the Hippo pathway20, emphasizing the importance of the Ex-Mer-Kib complex. Among all components of mammalian Hippo pathway, only NF2, the ortholog of Mer, undergoes heavy ubiquitin modification21. BRCA1/BARD1-mediated ubiquitination of NF2 does not promote its degradation, but rather influences its interactions with partners21. Moreover, NEDD4L-mediated Merlin ubiquitination on K396 promotes its binding to the downstream kinase Lats1, leading to the activation of the Hippo pathway22. Thus, ubiquitin modification on Mer always plays non-degradative roles. Due to the importance of Mer ubiquitination in the activation of the Hippo pathway, the mechanism regulating the activity of ubiquitin-modified Mer remains unclear but is crucial.
Ter94, also known as p97 or valosin-containing protein (VCP), is an evolutionarily conserved chaperone-like AAA+ ATPase that is widely expressed in eukaryotic cells23. It was initially identified for its involvement in endoplasmic reticulum-associated degradation24. Recent studies have gradually uncovered that Ter94 plays multiple roles in various cellular processes. One of its functions is the remodeling of chromatin through its ATPase activity, thereby facilitating DNA transcription25,26. Ter94 also participates in modulating RNA splicing and polyadenylation to control RNA metabolism27. Generally, Ter94 recognizes ubiquitin-modified proteins and extracts them for further processing28,29. The ATPase activity of Ter94 is dispensable for this extraction, as it requires energy to generate mechanical force30,31. In the cytoplasm, Ter94 specifically recognizes K11-linked ubiquitinated Ci, guiding it to proteasomes for partial degradation32. When cells are exposed to ultraviolet light, Ter94 facilitates the degradation of ubiquitin-modified XPC, triggering DNA damage33. In addition, Ter94 extracts K6-linked ubiquitinated c-MYC from the c-MYC-MAX heterodimer for subsequent proteasomal degradation34. Besides, Ter94 disassembles the PP1-SSD22-I3 inhibitory complex to activate PP1’s phosphatase activity, without affecting their protein levels35,36. Thus, Ter94 plays both degradative and non-degradative roles for ubiquitin-modified proteins.
In this study, we conducted an RNA interference (RNAi) screening in Drosophila, and identified that knockdown of Ter94 decreased wing size. Loss of Ter94 inhibited the expression of Hippo-responsive genes and triggered apoptosis in wing discs. In addition, we demonstrated that the AAA+ ATPase activity of Ter94 is crucial for its role in regulating the Hippo pathway. Interestingly, human VCP was able to functionally replace Ter94 in controlling wing size, Yki target gene expression, and apoptosis, reflecting their conservation. Through epistatic analyses, we determined that Ter94 located upstream of the core kinase cascade in modulating the Hippo pathway. Mechanistically, Ter94 bound Mer to weaken its interaction with Ex or Kib, without affecting the abundance of Mer. Thus, the disruption of the Ex-Mer-Kib complex by Ter94 leads to the suppression of the Hippo pathway. In summary, our study reveals that Ter94 suppresses the Hippo pathway by interfering with the formation of the Ex-Mer-Kib complex, thereby modulating wing size.
Results
Knockdown of ter94 decreases wing size
The Drosophila wing provides an ideal model for screening genes that determine organ size. To identify genes that control wing size, we crossed wing-specific nub-gal4 flies with RNAi lines to silence gene expression, and then observed wings of the offspring. Our unbiased screening revealed Ter94 as a potential regulator of wing size. Compared to the control RNAi (Fig. 1a), knockdown of ter94 using two RNAi lines from TsingHua Fly Center (1058) and Bloomington Drosophila Stock Center (35608) decreased wing size (Fig. 1b-c). The reduced wing size was specifically due to decreased ter94 expression, as introducing wild-type Ter94 was able to rescue the RNAi-induced phenotype (Fig. 1e-f). Interestingly, overexpressing wild-type Ter94 alone did not alter wing size (Fig. 1g versus Fig. 1d), suggesting that the endogenous Ter94 is sufficient to maintain proper wing size. Considering the ATPase activity of Ter94, we sought to examine whether its ATPase is involved in regulating wing size. In contrast to wild-type Ter94 (Fig. 1g), overexpression of a mutant form Ter94AA37,38, in which the ATP-binding sites (K248 and K521) were replaced by alanines, reduced wing size (Fig. 1h), suggesting a dominant-negative role of Ter94AA. Consistent with this view, co-expression of wild-type Ter94 to some extent rescued the small wing phenotype induced by Ter94AA (Fig. 1i). In line with this, previous studies have illustrated that Ter94AA plays a dominant-negative role in regulating the Hh32 and Notch pathways39, indicating that Ter94AA interferes with the endogenous Ter94 to exhibit a loss-of-function effect. To further confirm the role of Ter94 in wing size regulation, we employed the sd-gal4 driver to manipulate ter94 expression in wings. Compared to the control wing (Supplementary Fig. 1a), both knockdown of ter94 using 1058 (Supplementary Fig. 1b) or 35608 (Supplementary Fig. 1c) and overexpression of Ter94AA (Supplementary Fig. 1d) resulted in smaller wings. Knockdown of ter94 using THU3262 from TsingHua Fly Center led to adult lethality, so subsequent experiments on wing size focused on using 1058 and 35608 lines. Taken together, our genetic screening identified that Ter94 positively regulates wing size in an ATPase-dependent manner.
a–i Comparison of adult wings from control flies a, d, knockdown of ter94 by nub-gal4 b, c, overexpressing wild-type Ter94 g or Ter94AA h, Ter94 plus ter94 RNAi co-expression e, f, and Ter94 plus Ter94AA co-expression i. Quantification analyses were shown on right. The numbers in the bars represented the number of wings counted. Of note, knockdown of ter94 or overexpression of Ter94AA apparently diminishes wing size, which is restored by expressing Ter94. j A control wing disc expressing UAS-lacZ via sd-gal4 was stained to show Cas3 (white). k, l Wing discs with ter94 knockdown were stained to show Cas3. Notably, knockdown of ter94 is able to activate Caspase3. m Overexpression of Ter94AA elevated Cas3. n, p Overexpression of Ter94 rescued the upregulation of Cas3 induced by ter94 knockdown or Ter94AA. For all wing discs, red dotted lines mark the outlines of wing discs. Scale bars: 200 μm for all wings, 20 μm for all wing discs.
In fact, knockdown of Ter94 not only reduced wing size, but also caused wrinkles (Fig. 1b, c), resembling cell death. To investigate this further, we used the active-Caspase3 antibody for immunostaining. Compared to the control wing disc (Fig. 1j, Supplementary Fig. 2a), both ter94 knockdown (Fig. 1k, l, Supplementary Fig. 2b) and Ter94AA overexpression (Fig. 1m) triggered apoptosis, which could be recovered by co-expression of wild-type Ter94 (Fig. 1n, p, Supplementary Fig. 2c). On the other hand, BrdU incorporation assay showed that neither ter94 knockdown nor Ter94AA overexpression affected cell proliferation (Supplementary Figs. 1e–h). These findings suggest that depletion of Ter94 leads to reduced wing size, at least partially through the activation of apoptosis.
Loss of ter94 suppresses the expression of Yki target genes
Since the Hippo pathway plays an important role in regulating organ sizes, we tried to explore whether Ter94 is involved in this pathway. In general, the Hippo pathway exerts the pro-apoptotic effect via inhibiting the activity of its transcriptional co-factor Yki40. Thus, we utilized several well-characterized Yki readouts (diap1-lacZ, fj-lacZ and ban-lacZ) to evaluate the Hippo pathway activity. Compared to the control wing disc (Fig. 2a), knockdown of ter94 using 1058 or 35608 decreased diap1-lacZ levels (Fig. 2b, c). Additionally, overexpression of Ter94AA also downregulated diap1-lacZ (Fig. 2d), further supporting its dominant-negative effect. Similar results were obtained using another Yki readout, fj-lacZ (Fig. 2e–h). Since knockdown of ter94 using THU3262 driven by hh-gal4 causes lethality, so we used a temperature-sensitive ubiquitously expressed driver tub-Gal80ts to observe the change in fj-lacZ. As anticipated, knockdown of ter94 using THU3262 also reduced fj-lacZ level (Supplementary Fig. 2d). RT-qPCR analyses showed that all of these RNAi lines effectively silenced endogenous ter94 (Supplementary Fig. 2e). To validate these findings, we utilized a strong hypomorphic allele, ter94k15502, which contains a P-element insertion disrupting Ter94 expression32. Homozygosity for ter94k15502 was embryonic lethal, so we generated ter94k15502 homozygous clones in wing discs using the Flp recombinase/Flp recombinase target (FLP/FRT) method. Analysis of these clones, marked by the loss of green fluorescent protein (GFP) signals, revealed decreases in diap1-lacZ (Fig. 2i) and ban-lacZ (Fig. 2j). Consistent with the previous study39, the ter94k15502 homozygous clones exhibited reduced size, possibly due to decreased Yki activity. Furthermore, overexpression of wild-type Ter94 was able to restore the reductions in diap1-lacZ (Fig. 2k) and fj-lacZ (Fig. 2l) induced by 35608, as well as the decreases caused by Ter94AA (Fig. 2m, n). In summary, these results demonstrate that loss of ter94 suppresses the expression of Yki targets, and leads to growth disadvantage.
a–d Wing discs from control a, ter94 knockdown by en-gal4 b, c and Ter94AA overexpression via en-gal4 d were stained with Ci (red) and diap1-lacZ (white). The en-gal4 drives UAS transgenes to express in the posterior region of the wing disc, where does not express Ci. Both knockdown of ter94 and overexpression of Ter94AA decrease diap1-lacZ in wing discs. e-h Wing discs from control e, ter94 knockdown by hh-gal4 f, g and Ter94AA overexpression h were stained to show GFP (green) and fj-lacZ (white). GFP marks the expression pattern of hh-gal4. i-j Wing disc carrying ter94K15502 clones were stained to show the expression of GFP (green) and diap1-lacZ (white in i) or ban-lacZ (white in j). ter94K15502 clones are recognized by the lack of GFP. Of note, ter94 mutant cells exhibited decrease of diap1-lacZ (marked by arrows in i) and ban-lacZ (marked by arrows in j). k, l Wing discs simultaneously expressing V5-Ter94 and ter94 RNAi were stained to show V5 (red) and diap1-lacZ (white in k) or fj-lacZ (white in l). Overexpression of Ter94 enables to rescue the decrease of diap1-lacZ and fj-lacZ induced by ter94 knockdown. m, n Ter94 recovered the reduction of diap1-lacZ and fj-lacZ caused by Ter94AA. Scale bars: 20 μm for all wing discs.
Cytoplasmic hVCP protein enables to rescue ter94 RNAi-induced small wings
After demonstrating the necessity of Ter94 in maintaining proper wing size, we further investigated the functional conservation of Ter94/VCP. By conducting rescue assays with a human VCP transgenic fly, we found that the small wings caused by ter94-RNAi (Fig. 3a–c) were restored by expressing human VCP (Fig. 3e, f). Similar to wild-type Ter94, overexpression of hVCP alone did not impact wing size (Fig. 3d). These observations suggest that hVCP is able to substitute for Ter94 in modulating wing size.
a-f Comparison of adult wings from control flies a, knockdown of ter94 by nub-gal4 b, c, overexpressing human VCP d, and simultaneous expression of human VCP plus ter94 RNAi e, f. Quantification analyses were shown on right. The numbers in the bars represented the number of wings counted. Of note, small wings induced by ter94 knockdown are rescued by human VCP. g–o Comparison of adult wings from control flies g, expressing NES-hVCP h, expressing NLS-hVCP i, ter94 knockdown j, m, co-expression of ter94 RNAi plus NES-hVCP k, n, and co-expressing ter94 RNAi plus NLS-hVCP l, o. Quantification analyses were shown on right. The numbers in the bars represented the number of wings counted. Notably, small wings induced by ter94 knockdown are rescued by NES-hVCP, not by NLS-hVCP. Scale bars: 200 μm for all adult wings.
Previous studies have shown that Ter94 is involved in the degradation of both cytoplasmic and nuclear proteins through the proteasome pathway41. Immunostaining revealed that both V5-Ter94 and hVCP-V5 were present in the cytoplasm and nucleus (Supplementary Figs. 3a, b). To investigate whether cytoplasmic or nuclear Ter94 regulates wing size, we constructed transgenic flies expressing NES-hVCP and NLS-hVCP, which contain a nuclear export signal (NES) and a nuclear localization signal (NLS) respectively. Immunostaining confirmed that NES-hVCP-V5 exclusively resided in the cytoplasm (Supplementary Fig. 3c), while NLS-hVCP-V5 localized in the nucleus (Supplementary Fig. 3d). Overexpression of NES-hVCP or NLS-hVCP alone did not alter wing size (Fig. 3g–i). Remarkably, NES-hVCP successfully restored the small wings caused by ter94 knockdown (Fig. 3j, k, m, n), whereas NLS-hVCP did not (Fig. 3l, o), indicating that cytoplasmic Ter94 is important for regulating wing size.
Cytoplasmic hVCP rescues Yki activity suppression and apoptosis caused by ter94 knockdown
Given the above data showed that the Ter94/VCP plays a conserved role in regulating wing size, we sought to investigate whether VCP rescues ter94 RNAi-induced suppression of Yki target genes. Although overexpression of hVCP did not affect diap1-lacZ (Fig. 4a), it could restore the decreased diap1-lacZ caused by ter94 knockdown (Fig. 4b, c). In addition, NES-hVCP enabled to rescue ter94 RNAi-induced diap1-lacZ decreases (Fig. 3d–f), whereas NLS-hVCP failed to do so (Fig. 3g–i), together suggesting that cytoplasmic Ter94 plays a more important role in regulating the Hippo pathway.
a–c Wing discs expressing hVCP alone a, hVCP plus ter94 RNAi b, c were stained to show V5 (red) and diap1-lacZ (white). hVCP rescues diap1-lacZ expression under ter94 knockdown background. d-f Wing discs expressing NES-hVCP alone (d), NES-hVCP plus ter94 RNAi e, f were stained to show V5 (red) and diap1-lacZ (white). g–i Wing discs expressing NLS-hVCP alone g, NLS-hVCP plus ter94 RNAi h, i were stained to show V5 (red) and diap1-lacZ (white). j–o Wing discs expressing ter94 RNAi plus hVCP j, m, ter94 RNAi plus NES-hVCP k, n, or ter94 RNAi plus NLS-hVCP l, o were stained to reveal V5 (green) and Cas3 (white). As shown, the activation of Caspases by ter94 knockdown is rescued by NES-hVCP, not by NLS-hVCP. Scale bars: 20 μm for all wing discs.
After discovering that knockdown of ter94 triggers apoptosis, we proceeded to test whether hVCP could inhibit this process. The results showed that hVCP was able to block ter94 RNAi-induced apoptosis (Fig. 3j, m). Furthermore, cytoplasmic hVCP effectively inhibited the apoptosis caused by ter94 knockdown (Fig. 3k, n), while nuclear hVCP could not (Fig. 3l, o). Overall, Ter94/VCP plays a conserved role in regulating the Hippo pathway, and cytoplasmic Ter94 is important in this regulation.
Ter94 sits upstream of the core kinase cascade to control the Hippo pathway
Having demonstrated that loss of Ter94 decreases wing size and downregulates Yki target gene expression, we aimed to investigate the underlying mechanism. Central to the Hippo pathway is a kinase module, through which upstream signals converge on the transcriptional cofactor Yki, leading to the coordination of target gene expression42. By studying genetic interactions between Ter94 and key components of the Hippo pathway, we gained insight into how Ter94 modulates wing size. Because manipulating the Hippo pathway activity throughout the wing would result in deformation, we chose ptc-gal4 to drive transgene expression specifically between vein L3 and vein L443. Compared to the control wing (Fig. 5a), overexpression of Ter94AA using ptc-gal4 resulted in a noticeable decrease in the L3/L4 intervein size (Fig. 5b). Consistent with previous findings, overexpression of Yki or knockdown of Hippo pathway components, including mats, hpo, ex, kib and mer, increased the width of L3/L4 (Supplementary Fig. 4). Overexpression of Yki enabled the restoration of Ter94AA-induced undergrowth (Fig. 5c), indicating that Ter94 localizes upstream of Yki. This result further corroborated the notion that cytoplasmic Ter94 is more important for regulating the Hippo pathway. In addition, knockdown of core kinase module components, including mats and hpo also rescued the growth defect caused by Ter94AA (Fig. 5d, e), suggesting that Ter94 functions upstream of the core kinase cascade. We did not obtain a result for wts-RNAi since wts knockdown leads to larval lethality. Inhibition of the Ex-Mer-Kib branch only partially recovered the wing growth defect induced by Ter94AA (Fig. 5f–h), inferring that Ter94 may function in parallel with this complex.
a–h Comparison of adult wings from control a, expressing Ter94AA by ptc-gal4 b, simultaneously expressing Ter94AA plus Yki c, expressing Ter94AA plus mats RNAi d, expressing Ter94AA plus hpo RNAi e, expressing Ter94AA plus ex RNAi f, expressing Ter94AA plus kib RNAi g, and expressing Ter94AA plus mer RNAi h. The ptc-gal4 drives UAS transgenes to express between vein L3 and L4. Quantification analyses were shown below. The numbers in the bars represented the number of wings counted. Scale bars: 200 μm for all adult wings. i–k Wing discs expressing V5-Ter94AA alone by en-gal4 i, co-expressing V5-Ter94AA plus hpo RNAi j, expressing V5-Ter94AA plus wts RNAi k, and expressing V5-Ter94AA plus Yki l were stained to reveal V5 (red) and diap1-lacZ (white). Scale bars: 20 μm for all wing discs.
After observing that Ter94 is positioned upstream of the core kinase cascade in controlling wing size, we proceeded to assess Yki activity using diap1-lacZ as a readout. In comparison to the Ter94AA-overexpressing wing disc (Fig. 5i), simultaneous knockdown of hpo or wts successfully rescued the decreased diap1-lacZ (Fig. 5j-k). Furthermore, co-expression of Yki also restored Ter94AA-induced downregulation of diap1-lacZ (Fig. 5l). Taken together, these epistatic analyses indicate that Ter94 functions upstream of the core kinase cassette in regulating Yki activity.
Ter94 physically interacts with Mer
Previous studies have demonstrated that Ter94 primarily recognizes ubiquitin-modified proteins to deliver them to the proteasome for proteolysis. Given that our above results indicated that Ter94 likely acts in parallel to the Ex-Mer-Kib complex, we needed to examine the interaction between Ter94 and this complex. Three aspects point to Mer as the most likely binding partner of Ter94. First, co-immunoprecipitation (co-IP) and subsequent mass spectrometry analyses have revealed that Mer can pull down Ter9444. Additionally, compared to other components of the Hippo pathway, Mer exhibits dramatic ubiquitination21, which is a prerequisite for Ter94 recognition41. Finally, proteomic profiling of VCP substrates in mammalian cells indicates that Mer is a candidate34. Thus, we examined the interaction between Ter94 and Mer through co-IP assays. As expected, Myc-Mer reciprocally bound Fg-Ter94 (Fig. 6c-d). However, Yki or Hpo did not bind to Ter94 (Supplementary Figs. 5d-e), suggesting that Ter94 specifically interacts with Mer. Since the above findings demonstrate that human VCP can replace Ter94 in regulating the Hippo pathway and wing size, we tested whether human VCP binds to Mer or its human homolog NF2. The co-IP results displayed that hVCP interacts with Mer and NF2 (Supplementary Figs. 5a-c). Mer comprises a FERM domain in its N-terminus, which is important for mediating protein-protein interactions45. To explore whether the FERM domain is involved in Mer-Ter94 interaction, we generated a series of truncated mutants (Fig. 6a). The co-IP results revealed that Mer binds to Ter94 via its N-terminus (Fig. 6e), with the FERM domain being sufficient for this interaction (Fig. 6f). NF2 is a well-known tumor suppressor, with high-frequency mutations in its FERM domain. Several point mutations (L46R, F62S, L64P, L141P) have been shown to abolish the anti-tumor role of NF246. By sequence alignment, we strikingly found that these sites are conserved in Mer. Therefore, we mutated the corresponding sites and tested the interaction between these mutants and Ter94. As shown in Supplementary Fig. 5f, all mutants revealed weaker interactions with Ter94.
a, b The schematic diagrams show the domains of Mer and Ter94, and their truncated constructs used in the following co-IP. c Fg-Ter94 pulled down Myc-Mer in HEK-293T cells. d Myc-Mer pulled down Fg-Ter94 in HEK-293T cells. e Myc-Mer interacted with Fg-Ter94 through its N terminus. f The FERM domain in Mer enabled to pull down Fg-Ter94. g Both Fg-Ter94-N and Fg-Ter94-C were able to pull down Myc-Mer. h Fg-Ter94 and Fg-Ter94AA showed identical affinity to Myc-Mer. i Comparison of adult eyes from control (i-1), expressing Mer1-600 alone via GMR-gal4 (i-2), simultaneously expressing Mer1-600 and Ter94 (i-3). Quantification analyses of relative eye sizes were shown. The numbers in the bars represented the number of eyes counted. Scale bars: 100 μm for all adult eyes. j Comparison of adult wings from control (j-1), overexpression of Mer by nub-gal4 (j-2), co-expressing Mer plus Ter94 (j-3), expressing Mer1-600 alone (j-4), and co-expressing Mer1-600 plus Ter94 (j-5). Quantification analyses of relative wing size has been shown. The numbers in the bars represented the number of wings counted. Scale bars: 200 μm for all adult wings.
On the other hand, to map the fragment of Ter94 responsible for binding Mer, we constructed three nonoverlapping truncated mutants (Fig. 6b). The co-IP assays showed that both the N-terminus and C-terminus of Ter94 were able to interact with Mer, while the ATPase domains failed to bind Mer (Fig. 6g). Furthermore, Ter94 and Ter94AA exhibited equivalent affinities for Mer (Fig. 6h), providing an explanation as to why Ter94AA plays a dominant-negative role.
Having demonstrated the interaction between Mer and Ter94, we next explored whether Ter94 regulates the anti-growth activity of Mer. It is known that wild-type Mer forms an auto-inhibitory structure, that can be relieved by deleting its C-terminal 35 amino acids47. Ectopic expression of Mer1-600 by GMR-gal4 slightly decreased eye size and led to roughness, which was recovered by co-expression of Ter94 (Fig. 6i). Similarly, overexpression of Mer using nub-gal4 mildly decreased wing size, which was rescued by co-expressing Ter94 (Fig. 6j). In line with the previous finding47, Mer1-600 overexpression resulted in smaller wings, but this effect was restored by co-expression of Ter94 (Fig. 6j). These results indicate that Ter94 has the ability to suppress Mer activity.
Given that Ter94 primarily directs proteins to proteasome-mediated proteolysis23,48, it was necessary to test whether Ter94 promotes Mer degradation. Due to the unavailability of a commercial Mer antibody, we generated a tub-Myc-Mer transgenic fly that expressed Myc-tagged Mer protein under the tubulin promoter. The Myc antibody was used to detect Myc-Mer protein levels, which were found to be evenly expressed in the wing disc (Supplementary Fig. 6a). Overexpression of mer RNAi was able to diminish Myc-Mer protein, confirming the reliability of the tub-Myc-Mer fly (Supplementary Fig. 6b). Surprisingly, knockdown of ter94 did not impact Myc-Mer protein levels (Supplementary Figs. 6c-d), and overexpression of Ter94AA (Supplementary Fig. 6e) or wild-type Ter94 (Supplementary Fig. 6f) also had no effect. Previous studies have demonstrated the importance of the apical localization of Mer in activating the Hippo pathway49. Therefore, we investigated whether Ter94 influences the localization of Mer. While Mer is typically found colocalizing with the apical domain marker Dlg (Supplementary Fig. 6g), overexpression of Ter94 resulted in a decrease in the apical positioning of Mer within epithelial cells (Supplementary Fig. 6h). In sum, Ter94 binds to the FERM domain of Mer to suppress its activity, without affecting its protein abundance.
Ter94 dissociates the Ex-Mer-Kib complex
Previous studies have demonstrated that Mer forms a complex with Ex and Kibra at the apical domain of cells to recruit Wts for phosphorylation, ultimately activating the Hippo pathway20,47,50. In view of the interaction between Ter94 and Mer, we attempted to investigate whether Ter94 interferes with the formation of Mer-containing complexes. Mer1-600 recruits Wts to the cell membrane via its N-terminal FERM domain, resulting in Wts phosphorylation and subsequent activation47. Interestingly, our results showed that co-expression of Ter94 decreased the interaction between Mer1-600 and Wts (Fig. 7a). In addition, we observed that Ter94 was able to inhibit the binding of Mer to Ex (Fig. 7b) and Kib (Fig. 7c). Since the formation of an Ex-Mer-Kib complex is crucial for activating the Hippo pathway18, our findings suggest that Ter94 may suppress the pathway by dissociating this complex.
a HA-Ter94 decreased the interaction between Fg-Mer1-600 and Myc-Wts. b HA-Ter94 suppressed Fg-Mer binding to Myc-Ex. c HA-Ter94 diminished the interaction between Fg-Kib and Myc-Mer. d Co-transfection of Ub-K63 promoted Mer binding to Ex. e Co-expression of Ub-K63 enhanced the interaction between Kib and Mer. f A proposed model of Ter94 inhibiting the Hippo pathway. Ter94 recognized K63-linked ubiquitinated Mer to inhibit it from forming the Ex-Mer-Kib complex, thereby suppressing the initiation of the Hippo pathway.
Several previous studies have revealed that Mer undergoes non-degradative polyubiquitination, which alters its interactions with partners21,22. As K63-linked polyubiquitination is always involved in the regulation of protein-protein interactions, we explored whether this modification affects the assembly of the Ex-Mer-Kib complex. To eliminate the influence of proteolysis caused by ubiquitination, we opted for the Ub-K63 mutant. This mutant replaces all lysines (Ks) except K63 in ubiquitin with arginines, leaving only K63 to form K63-linked polyubiquitin chains. Remarkably, co-transfection of Ub-K63 enhanced the interaction between Mer and Ex (Fig. 7d) as well as Kib (Fig. 7e), suggesting that ubiquitinated Mer prefers to form a complex with Ex and Kib. In conclusion, our study proposes a possible mechanism in which Ter94 recognizes ubiquitinated Mer and prevents it from forming the Ex-Mer-Kib complex, leading to inactivation of the Hippo pathway (Fig. 7f).
Discussion
Organ size determination is a complex and interesting biological process that is regulated by multiple mechanisms, with the Hippo pathway playing a key role. The Hippo pathway was initially discovered in Drosophila through mutagenesis screening51. Mutation of several components of this pathway leads to organ overgrowth16. Central to the pathway is the Hpo-Wts kinase module, which is activated by the upstream Ex-Mer-Kib complex19. Previous studies have revealed that ubiquitin modification on Mer is an important step for its activation, rather than leading to its degradation21,22. However, the mechanism for terminating the activity of ubiquitinated Mer has remained elusive. In this study, through genetic screening, we identified that Ter94 positively regulates wing size dependent on its ATPase activity. Knockdown of ter94 decreased wing size, and downregulated the expression of Yki target genes. Human VCP was able to restore ter94 RNAi-induced growth defect and Yki target gene inhibition. Furthermore, cytoplasmic Ter94 was more important for regulating wing size and the Hippo pathway. Based on epistatic analyses, we fingered out that Ter94 acts in parallel with the Ex-Mer-Kib complex to modulate the Hippo pathway. Mechanistically, Ter94 recognized the ubiquitinated Mer to prevent it from forming the Ex-Mer-Kib complex, thereby suppressing the Hippo pathway. This study reveals a mechanism to cease the activity of ubiquitinated Mer without promoting its proteolysis.
Although knockdown of ter94 decreases wing size, and depletion of ter94 leads to growth disadvantage, overexpression of Ter94 does not lead to an increase in wing size or activation of Yki target genes. These observations can be attributed to two reasons. First, endogenous Ter94 is sufficient to regulate Mer and maintain the normal activity of the Hippo pathway. Consistent with this possibility, overexpression of Ter94 indeed rescues the small wing and eye induced by Mer1-600. Alternatively, only a small portion of Mer is subject to ubiquitin modification, a key requirement for Ter94 recognition. A previous study showed that Ter94 prefers to bind K11-linked polyubiquitinated Ci32, while another study found that Ter94/VCP recognizes K6-linked polyubiquitinated c-MYC34. In this study, Ter94 possibly binds to K63-linked polyubiquitin chains attached to Mer. Hence, Ter94 is able to recognize distinct polyubiquitin linkages depending on different substrates. In contrast to the canonical role of Ter94, it fails to modulate the stability of Mer. How Ter94 coordinates its degradative and non-degradative roles on different substrates will be an interesting research direction.
Mer is a renowned tumor suppressor, as its somatic mutations have been tightly linked to the development of several types of tumors, particularly schwannomas and meningiomas52,53. A meta-analysis has revealed that most tumor-derived Mer mutations cluster in its FERM domain54, which is responsible for binding Ter94. It would be beneficial to investigate whether these mutations disrupt Mer binding to Ter94, thereby relieving the inhibitory effect of Ter94. As a matter of fact, inhibition of Ter94/VCP is considered to be a promising strategy for tumor intervention55,56. Several Ter94/VCP inhibitors, including CB-5083 and CB-5339 are under clinical trials57. In the further, it is necessary to explore whether Ter94/VCP inhibitors exert anti-tumor effects by activating the Hippo pathway. Thus, this study facilitates to dissect the mechanism of Ter94/VCP inhibitors inhibiting tumor progression and provides guidance for their clinical application.
Materials and methods
Drosophila genetics
nub-gal4, en-gal4, hh-gal4, sd-gal4, ptc-gal4, GMR-gal4, ap-gal4, hpo-RNAi, wts-RNAi, UAS-Yki, UAS-lacZ, diap1-lacZ, ban-lacZ and fj-lacZ have been described in our previous studies14,58,59,60. ter94-RNAi (1058, THU3262), mats-RNAi (THU3571), hpo-RNAi (THU0551), ex-RNAi (TH201501137.S), kib-RNAi (THU3065), mer-RNAi (THU2845) were purchased from TsingHua Fly Center (THFC). ter94-RNAi (35608), tub-gal80ts were obtained from Bloomington Drosophila Stock Center (BDSC). UAS-V5-Ter94, UAS-V5-Ter94AA, UAS-hVCP-V5, UAS-NES-hVCP-V5, UAS-NLS-hVCP-V5 transgenic flies were purchased from Core Facility of Drosophila Resource and Technology, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. UAS-HA-Mer and UAS-HA-Mer1-600 transgenic flies were kindly from Prof. Shian Wu, Nankai University. The attB-tub-Myc-Mer construct was made by cloning a full-length mer cDNA inserted into downstream of the α-tubulin promoter, then inserting this construct into 25C6 attP locus (#25709, BDSC)59.
DNA constructs
To generate Myc-Mer, Fg-hVCP, Myc-NF2, Myc-Yki, HA-Hpo, Myc-Wts, Myc-Ex, Fg-Kib, Fg-Ter94 and HA-Ter94 constructs, we amplified the corresponding cDNA fragments using Vazyme DNA polymerase (P505), and inserted them into pcDNA3.1-Myc, pcDNA3.1-Fg or pcDNA3.1-HA backbone vectors respectively. Truncated constructs including Myc-Mer-N (aa1-375), Myc-Mer-C (aa376-635), Myc-Mer-N1 (aa1-350), Myc-Mer-N2 (aa1-325), Myc-Mer-N3 (aa1-300), Fg-Mer1-600, Fg-Ter94-N (aa1-210), Fg-Ter94-M (aa211-660), and Fg-Ter94-C (aa661-801) were made by inserting the corresponding coding sequences into pcDNA3.1-Myc or pcDNA3.1-Fg vectors. Fg-Ter94AA, Myc-Mer-L36R, Myc-Mer-F52S, Myc-Mer-L54P, Myc-Mer-L135P and HA-Ub-K63 were made by PCR-based site-directed mutagenesis.
Immunostaining and confocal
Immunostaining of wing discs was carried out according to our previous protocols61. Briefly, third-instar larvae were dissected in PBS and fixed in 4% PFA at room temperature for 20 min, then permeabilized with PBT (PBS supplemented with 0.1% Triton X-100) for three times. Larvae were incubated with primary antibodies in PBT at 4 °C for at least 4 hr, then washed with PBT for three times and incubated with fluorophore-conjugated secondary antibodies for 2 hr at room temperature. After washing for three times with PBT, discs were separated and mounted with 40% glycerol. Images were captured by Zeiss confocal microscope. Primary antibodies used in this study included: mouse anti-V5 (1:500, MBL, M215-3), rabbit anti-cleaved Caspase-3 (1:200, Cell Signaling Technology, 9661 S), rabbit anti-β-Galactosidase (1:500, MBL, PM049), mouse anti-Myc (1:200, Santa Cruz, sc-40), rat anti-Ci (1:10, DSHB, 2A1), mouse anti-Dlg (1:10, DSHB, 4F3), rat anti-HA (1:200, Santa Cruz, sc-53516). To mark cell nuclei, wing discs were stained with DAPI (1:10000, Santa Cruz, sc-24941) for 15 min before mounting. All secondary antibodies used in this study were bought from Jackson ImmunoResearch, and were diluted at 1:500.
BrdU labeling
Wing discs were incubated with 30 μM BrdU (Sigma, HY-15910) for 45 min in S2 medium (Hyclone) before fixation, and the subsequent immunostaining was performed according the standard protocol. Primary antibodies used in this study was mouse anti-BrdU (1:10, DSHB, G3G4).
RNA isolation, reverse transcription, and real-time PCR
Wing discs for ter94-RNAi (1058, 35608, THU3262) driven by nub-gal4 lysed in TRIzol (Invitrogen) for RNA isolation following standard protocols. 500 ng RNA were used for reverse transcription by MonScriptTM product line (Monad) according to the instructions. Real-time PCR was performed on ZY/VQ-100A (Yuanzan) using the ChamQ SYBR qPCR Master Mix (Q711, Vazyme). 2-ΔΔCt method was used for relative quantification. The primer pairs used was follows: ter94, 5’-AAG CTG GCC ATC CGA CAG-3’ (forward), 5’-ATG GCC TCC TCG AAG TGG G-3’ (reverse); actin, 5’-GTA CCC CAT TGA GCA CGG TA-3’ (forward) and 5’-ACT CCT GCT TGC TGA TCC AC-3’ (reverse). All RT-qPCR results are presented as means ± SD (standard deviation) of values from at least three experiments.
Cell culture, transfection, and immunoblot
All cell-based assays in this study were carried out in HEK-293T cells. HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM). Transfection was performed using PEI (Sigma) according to the manufacturer’s instructions. 48 h after transfection, cells were collected for subsequent co-IP and IB according to our previous described62. The following antibodies were used for IP and IB: mouse anti-Fg (1:500 for IP, 1:5000 for IB, Sigma, F3165); mouse anti-Myc (1:200 for IP, 1:2000 for IB, Santa Cruz, sc-40); mouse anti-HA (1:2000 for IB, Santa Cruz, sc-7392); goat anti-mouse HRP (1:10000, Abmax). Uncropped blots are shown in Supplementary Fig. 7 and Supplementary Fig. 8.
Statistics and reproducibility
Sizes of wings and eyes were measured by Image J software. Statistical analyses were performed with GraphPad Prism software, using one-way ANOVA. All data were presented as means ± SD (standard deviation), and P < 0.05 was considered statistically significant. Quantitative analyses were shown, with the numbers in the bars indicating the number of wings and eyes that were counted. All source data underlying the graphs are presented in Supplementary Data. All wing disc images were captured by selecting three images with consistent trends of change. The WB data shown in the article are representative and have been repeated three times.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The numerical source data behind the graphs can be found in the Supplementary data file. All other data are available from the corresponding author on reasonable request.
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Acknowledgements
We sincerely thank Dr. Dezhen Peng, the founder of Yuanzan Lifescience Co., Ltd, for donating a quantitative PCR instrument (ZY/VQ-100A) to our lab. We also appreciate Prof. Shian Wu (Nankai University, China) and Prof. Junzheng Zhang (China Agricultural University, China) for generous providing Drosophila stocks. We also appreciate Shanghai Institute of Biochemistry and Cell Biology, Bloomington Drosophila Stock Center (BDSC) and TsingHua Fly Center for providing flies, and Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa for providing antibodies. This study was supported by grants from the National Natural Science Foundation of China (32270522, 32272945 and 32350710192) and the project of Double Thousand Plan in Jiangxi Province of China (09030049).
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The authors have made the following declarations about their contributions: Z.Z. and Q.L. designed the experiments. M.L., W.D., Y.D. and Y.Z. performed the experiments. Z.Z. and Q.L. carried out data analysis. M.L. and Z.Z. wrote the manuscript with the help of all authors.
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Li, M., Ding, W., Deng, Y. et al. The AAA-ATPase Ter94 regulates wing size in Drosophila by suppressing the Hippo pathway. Commun Biol 7, 533 (2024). https://doi.org/10.1038/s42003-024-06246-x
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DOI: https://doi.org/10.1038/s42003-024-06246-x
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