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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2024 Jul 1;300(8):107512. doi: 10.1016/j.jbc.2024.107512

NEDD8 enhances Hippo signaling by mediating YAP1 neddylation

Mengjuan Chen 1, Yuqing Liu 1, Mingzhong Zuo 1, Chaohui Guo 1, Yongkun Du 2, Huifen Xu 1, Bianzhi Liu 1, Ming Li 1, Wuhan Xiao 3,4,5,∗, Guangqing Yu 1,∗
PMCID: PMC11327456  PMID: 38960037

Abstract

The Hippo–YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo–YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling.

Keywords: NEDD8, YAP1, neddylation, Hippo signaling, GCs apoptosis

The Hippo signaling pathway is highly conserved and controls organ growth and size, cell proliferation, regeneration, migration, and apoptosis (1, 2, 3, 4). The mammalian Hippo signaling pathway comprises two kinases (LATS1/2 and MST1/2) and two transcriptional coactivators (yes-associated protein 1 (YAP1) and TAZ). Upon activation by upstream signals, LATS1/2 kinases are phosphorylated by MST1/2 (5, 6). Activated LATS1/2 then phosphorylated YAP1 and TAZ, preventing YAP1 from entering the nucleus and leading to its proteasomal degradation in the cytoplasm (7). However, the unphosphorylated YAP1 protein can translocate into the nucleus to enhance TEA domain-dependent gene transcription and induce downstream gene expression (8).

Neddylation is a posttranslational modification (PTM) involving the covalent attachment of Nedd8 to substrates in a ubiquitin-like manner, which plays an important role in various biological processes such as cell cycle progression, signal transduction, and immune recognition (9, 10). The neddylation system consists of the Nedd8-activating enzyme E1 (NAE, a heterologous dimer consisting of UBA3 and NAE1/APP-BP1), the Nedd8-conjugating enzyme E2 (UBC12), and substrate-specific E3 ligases (11, 12). The activated Nedd8 forms a covalent conjugate with the lysine residue of the target proteins at its carboxy-terminal glycine 76, resulting in the generation of different types of neddylation chains (13, 14). MLN4924, a small molecule-specific inhibitor of NAE (15, 16), is widely used to inhibit Nedd8 function.

Accumulating evidence highlights the essential role of PTMs in the regulation of the Hippo–YAP signaling pathway. Therefore, we further investigated whether and how neddylation regulates the YAP-mediated signaling pathway. Here, we found that NEDD8 activates Hippo-YAP signaling by mediating YAP1 neddylation. Furthermore, inhibition of neddylation leads to inhibition of YAP1 transcriptional activity and a decrease in YAP1 accumulation in the nuclei of granulosa cells (GCs). Further assays indicated that xiap may act as a potential NEDD8 E3 ligase of YAP1 to promote its neddylation.

Results

Expression and localization of rabbit NEDD8 in the ovarian follicles

Our previous study has shown that loss of nedd8 in zebrafish results in ovarian developmental defects, oocyte arrest at early developmental stages, and altered locomotor activity in males, suggesting that nedd8 plays a critical role in vivo (17). To identify NEDD8 expression patterns in rabbits, we first compared the homology of the human NEDD8 protein, which is evolutionarily conserved across the three species (Fig. 1A). Phylogenetic analysis confirmed NEDD8 phylogenetic relationships among different species (Fig. 1B). Structural prediction showed that the primary configuration of NEDD8 is conserved between rabbit NEDD8 and zebrafish nedd8 (Fig. 1C) (18). The mRNA expression levels of NEDD8 are higher in the muscle, heart, and ovary than other tissues in rabbits (Fig. 1D). As shown in Figure 1, E and F, NEDD8 is predominantly localized in follicles at different developmental stages and in the nucleus of GCs.

Figure 1.

Figure 1

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Phylogenetic analysis of NEDD8 proteins and the expression profile of rabbit NEDD8 in different tissues.A, alignment of NEDD8 amino acid sequences from Homo sapiens (Hs), Oryctolagus cuniculus (Oc), and Danio rerio (Dr). B, phylogenetic tree of NEDD8 proteins in 11 species constructed using the neighbor-joining method. Homo sapiens (Hs), Gene ID: 4738; Oryctolagus cuniculus (Oc), Gene ID: 100009008; Mus musculus (Ms), Gene ID: 18002; Sus scrofa (Ss), Gene ID: 100520086; Gallus gallus (Gg), Gene ID: 100858776; Latimeria chalumnae (Lc), Gene ID: 102345978; Xenopus tropicalis (Xt), Gene ID: 549727; Danio rerio (Dr), Gene ID: 368667; Oryzias latipes (Ol), Gene ID: 101172934; Takifugu rubripes (Tr), Gene ID: 101077379; Drosophila melanogaster (Dm), Gene ID: 35151. C, predicted protein structure of rabbit NEDD8 by UniProtKB in the Ensemble database (https://www.uniprot.org/uniprotkb/Q4PLJ0/entry). D, mRNA expression levels of NEDD8 in various tissues of adult rabbits. Data are expressed as mean ± SD, n = 3 experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. E, immunofluorescence staining of NEDD8 in follicles at different developmental stages in rabbit ovaries. The scale bar represents 20 μm and 100 μm, respectively. F, confocal microscopy image of endogenous NEDD8 localization in rabbit GCs. NEDD8 is stained red and nuclei are stained blue with DAPI. The scale bar represents 50 μm. DAPI, 4′, 6-diamidino-2-phenylindole; EV, early vitellogenic stage; FG, full-grown stage; GC, granulosa cell; MV, mid-vitellogenic stage; ns, not significant; PG, primary growth stage; PV, previtellogenic stage.

Hippo signaling is significantly enriched in MLN4924-treated GCs

Previously, we used transcriptomic analysis to investigate the gene transcription profile of rabbit GCs after treatment with MLN4924 (19). In this study, we present a subset of the signaling pathways enriched by differentially expressed genes (DEGs), including oncogenic, cell cycle, PI3K-Akt, and Hippo pathways (Fig. 2A). Among the identified signaling pathways, we focused on Hippo-YAP signaling because of its critical involvement in various ovarian biological processes. We hypothesized that neddylation could modulate YAP1 signaling. The upregulated and downregulated genes in the Hippo signaling pathway are shown in Figure 2B. Among them, the downregulation of GLI2 and ALIN2, which are associated with proproliferation, belong to YAP1 target genes. In addition, WNT5A and WNT2 are upregulated, while WNT10B and WNT8A are downregulated. These genes are components of the Wnt signaling pathway, which are critical for cell proliferation and differentiation. The trend of these DEGs in nedd8−/− zebrafish is consistent with the above results (Fig. 2C). Taken together, these data support that inhibition of neddylation can negatively regulate the expression of genes in the Hippo signaling pathway.

Figure 2.

Figure 2

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Bioinformatic analysis of differentially expressed genes in rabbit ovarian GCs treated with DMSO or MLN4924.A, representative KEGG enrichment results of differentially expressed genes between rabbit ovarian GCs of DMSO and MLN4924 treatment, respectively. The data point size indicates the number of enriched genes. | log2 FoldChange | >1.5 and p value <0.05 are set as screening criteria. B, expression heatmap of DEGs in the Hippo pathway. Red represents increased gene expression, while blue represents decreased gene expression. The color legend shows the FPKM values each represents. C, qPCR analysis of DEGs related to Hippo pathway in zebrafish. Data are expressed as mean ± SD, n = 3 experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, using unpaired Student’s t test. DEG, differentially expressed gene; DMSO, dimethyl sulfoxide; FPKM, fragments per kilobase million; GC, granulosa cell; KEGG, Kyoto encyclopedia of genes and genomes; ns, not significant; qPCR, quantitative real-time PCR.

NEDD8 promotes the transcriptional activity of YAP1

To further determine whether neddylation could indeed modulate the Hippo signaling pathway, we sought to investigate the effect of NEDD8 on the pathway. First, promoter assays were used to assess the effect of NEDD8 on YAP1 transcriptional activity. The 8xGTIIC-luciferase, a YAP/TAZ luciferase reporter (20), was used to monitor rabbit YAP1 activity. Overexpression of YAP1 in HEK293T cells, a human kidney cell line widely used for reporter assays and immunoblotting (20, 21), activated the promoter activity (Fig. 3A). When NEDD8 was overexpressed together with YAP1, the 8xGTIIC-luciferase promoter activity increased dramatically (Fig. 3A). Conversely, the promoter activity induced by YAP1 overexpression was decreased after MLN4924 treatment (Fig. 3B). Consistent with these findings, quantitive real-time PCR analysis showed that YAP1-activated genes (Birc5, CCNB1, CDC20, CTGF, CCND1) were downregulated and YAP1-repressed genes (SFN, GADD45A, CDKN1A) were upregulated in MLN4924-treated GCs (Fig. 3C). Taken together, these data suggest that NEDD8 may act as a positive regulator of the YAP signaling pathway.

Figure 3.

Figure 3

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Neddylation enhances YAP-mediated signaling.A, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-YAP1 (200 ng) transfected HEK293T cells with or without HA-NEDD8 for 36 h. B, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-YAP1 (200 ng) transfected HEK293T cells with or without MLN4924 (1 μM) treatment for 24 h. C, quantitative real-time PCR (qPCR) analysis of genes either positively or negatively regulated by YAP1 in rabbit GCs treated with or without MLN4924 (1 μM) for 24 h. Four separate experiments were performed, with each measurement performed at least three times. Data are expressed as mean ± SD. (unpaired two-tailed Student's t test). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. GC, granulosa cell; ns, not significant; YAP1, yes-associated protein 1.

Neddylation inhibition promotes YAP1 phosphorylation and reduction of its nuclear accumulation

As previously reported, NEDD8-substricted cullin 7 enhances the ubiquitination and degradation of Mst1, thereby activating the YAP signaling pathway in the heart development (22). Next, we investigated whether the effect of neddylation on YAP1 is related to the previously described regulation of YAP1 by the Hippo pathway. Immunofluorescence staining confirmed the colocalization of YAP1 and MST1 with NEDD8 in rabbit ovaries (Fig. 4, A and B). Furthermore, we performed colocalization assays by overexpressing EGFP-NEDD8 and mCherry-YAP1 in H1299 cells. The subcellular localization of YAP1 and NEDD8 provides valuable insights into their potential roles in cellular responses. Activation of the Hippo signaling pathway results in the phosphorylation of YAP1, causing its translocation from the nucleus to the cytoplasm, thereby inhibiting its function as a transcriptional coactivator (23). As shown in Figure 4C, NEDD8, indicated by green signal, partially overlapped with YAP1. MLN4924 treatment increased the protein levels of MST1 and phospho-YAP1, while decreasing YAP1 and YAP1 target genes (CDKN1A and CTGF) (Fig. 4, D and E). These data suggest that neddylation enhances the activity of the YAP-mediated signaling.

Figure 4.

Figure 4

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Neddylation of YAP1 promotes its nuclear translocation.A and B, immunofluorescence staining of endogenous NEDD8 and MST1 (A), NEDD8 and YAP1 (B) expression, and localization in the rabbit ovary. The nucleus was stained blue with DAPI (the scale bar represents 100 μm). C, confocal microscopy image of cotransfection with EGFP-NEDD8 and mCherry-YAP1 plasmids in H1299 cells, the nucleus was stained blue with DAPI (the scale bar represents 10 μm). D, Western blot analysis of the indicated protein levels of YAP1, p-YAP1, NEDD8, CDKN1A, MST1, and CTGF in rabbit GCs treated with or without MLN4924 (1 μM) for 0 to 48 h. E, quantification of the above proteins expression level. F, neddylation of YAP1 is detected by immunoprecipitation and Western blot in HEK293T cells in response to NEDD8 overexpression using Ni-NTA agarose beads. G, Western blot analysis and the quantification of the indicated protein in nuclear (Nuc) versus cytoplasmic (Cyt) fractionation from rabbit GCs with or without MLN4924 (1 μM) treatment for 24 h. Data are represented as means ± SD, n = 3 experiments, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Cyt, cytoplasm; DAPI, 4′, 6-diamidino-2-phenylindole; GC, granulosa cell; IB, immunoblotting; IP, immunoprecipitation; ns, not significant; Nuc, nucleus; TCL, total cell lysate; YAP1, yes-associated protein 1.

To further determine whether YAP1 is also a target for neddylation, we first performed in vitro neddylation assays. Coexpression of NEDD8 and YAP1 resulted in distinct NEDD8-conjugated YAP1 bands (Fig. 4F). Strikingly, after treatment with MLN4924, the protein level of YAP1 was decreased in the nuclei and the phosphor-YAP1 was increased in the cytoplasm (Fig. 4G). Therefore, these data suggest that YAP1 neddylation promotes its nuclear translocation and enhances the Hippo–YAP signaling pathway.

Inhibition of YAP signaling stimulates MLN4924-induced GCs apoptosis

Inhibition of neddylation by MLN4924 has been shown to increase apoptosis in rabbit GCs (19), with phenotypes similar to those observed in human YAP1-deficient GCs (24). To investigate whether the beneficial effects of MLN4924 on GCs apoptosis are mediated by YAP1 downregulation, we first suppressed YAP signaling in rabbit GCs using verteporfin (VP) and confirmed the efficiency of YAP1 and its target protein suppression by Western blotting. Our results showed a gradual decrease in the protein levels of YAP1 and Birc5, with 5 μM selected as the optimal concentration of VP (Fig. 5, A–C). Changes in YAP1 target gene expression after VP and MLN4924 treatments were consistent. Subsequently, we treated GCs with VP in combination with MLN4924, which resulted in a more significant decrease in the mRNA and protein expression levels of the YAP1-activated target genes CTGF and Birc5 (Fig. 5, D, E, H and I), while the mRNA levels of YAP1-inhibited target genes, SFN and GADD45A, showed a more pronounced increase (Fig. 5, F and G). In addition, cotreatment with VP and MLN4924 resulted in significantly enhanced expression of proapoptotic marker genes compared to single treatments (Fig. 6, A, B, D–F), along with significant suppression of antiapoptotic marker gene expression (Fig. 6C). Finally, to validate these findings, Caspase3 immunofluorescence was performed, which revealed a significant increase in Caspase3 in cells treated with both VP and MLN4924 compared to their individual treatments (Fig. 6G). These results support the hypothesis that inhibition of YAP signaling stimulates MLN4924-induced apoptosis of GCs.

Figure 5.

Figure 5

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MLN4924 and VP work together to inhibit YAP1-targeted gene expression of GCs.A–C, GCs were treated with VP at various doses (0, 0.1, 0.5, 1, 5, 10 μM) for 24 h. Cell lysates were subjected to Western blotting, and the protein levels of YAP1 and Birc5 were quantified and normalized to that of β-actin. A, representative experiment from three separate experiments is shown. B, quantification of YAP1 protein expression level in VP-treated cells. C, quantification of Birc5 protein expression level in VP-treated cells. D–G, qPCR analysis of YAP1-activated target genes Birc5 (D) and CTGF (E) mRNA levels, and YAP1-inhibited target genes SFN (F) and GADD45A (G) mRNA levels in GCs treated with DMSO, ML4924 (1 μM), VP (5 μM), or ML4924 and VP together for 24 h. H–J, GCs were treated with DMSO, ML4924, VP, or ML4924 and VP together for 24 h. Cell lysates were used for Western blot, and CTGF and Birc5 protein levels were subsequently quantified and normalized to that of β-actin. A representative Western blot (H), the quantification of CTGF (I) and Birc5 (J) from three independent experiments. Data are presented as means ± SD, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. DMSO, dimethyl sulfoxide; GC, granulosa cell; ns, not significant; qPCR, quantitative real-time PCR; VP, verteporfin; YAP1, yes-associated protein 1.

Figure 6.

Figure 6

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Effect of YAP1 suppression on MLN4924-induced apoptosis in rabbit GCs.A–C, qPCR analysis of proapoptotic marker genes Bax (A), Caspase3 (B), and antiapoptotic marker gene Bcl2 (C) mRNA levels in GCs treated with DMSO, MLN4924 (1 μM), VP (1 μM), or MLN4924 and VP together for 24 h. D–F, GCs were treated with DMSO, MLN4924 (1 μM), VP (5 μM), or MLN4924 and VP together for 24 h. Cell lysates were used for Western blot, and then YAP1 and Caspase3 protein levels were quantified and normalized to that of β-actin. A representative Western blot (D), the quantification of YAP1 (E) and Caspase3 (F) from three independent experiments. G, representative immunofluorescence staining for Caspase3 (red) after DMSO, MLN4924 (1 μM), VP (5 μM), or MLN4924 and VP together for 48 h. Quantification analysis of relative Caspase3 expression level (fluorescence intensity of Caspase3/DAPI) by Image J software. Data are presented as mean ± SD, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. DAPI, 4′, 6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; GC, granulosa cell; ns, not significant; qPCR, quantitative real-time PCR; VP, verteporfin; YAP1, yes-associated protein 1.

Disruption of nedd8 in zebrafish results in repression of the Hippo–YAP signaling pathway

To validate the effect of neddylation on the YAP signaling pathway in vivo, the mRNA expression levels of YAP1 targets, including both activated and repressed genes, were examined in the nedd8-null zebrafish (Fig. 7A) ovary, brain, and muscle. As expected, nedd8 disruption significantly suppressed yap signaling, including the downregulation of yap1-activated genes (ctgf, ccnb1, cdc20, areg, ccna2, aukb, gli2, cyr61, auka) and upregulation of yap1-repressed genes (gadd45a) expression (Fig. 7, B–D). Furthermore, Western blot analysis revealed decreased protein levels of yap1 and yap1 target activated proteins (ctgf and birc5) in the nedd8-null zebrafish brain (Fig. 7E). Taken together, these data suggest that disruption of nedd8 in zebrafish suppresses the YAP-mediated signaling.

Figure 7.

Figure 7

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Disruption of nedd8 in zebrafish results in the inhibition of yap1 signaling.A, sequence information diagram of nedd8-null zebrafish. A 4-bp nucleotide deletion (5′-GAGC-3′) occurred in exon 3 of the nedd8 deletion mutant, resulting in a reading frame shift. B–D, qPCR analysis of yap1 target genes that are either positively or negatively regulated at the mRNA level in WT and nedd8-null zebrafish ovary (B), brain (C), and muscle (D). β-actin was used as an internal control. E, Western blot analysis of GAPDH, birc5, ctgf, yap1, and nedd8 protein levels in brain of nedd8+/+ and nedd8−/− zebrafish. GAPDH was used as an internal control. The red numbers represent the quantified results of the protein. Data are representative of three independent experiments (mean ± SD of three technical replicates). Unpaired two-tailed Student's t test was performed. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. ns, not significant; qPCR, quantitative real-time PCR; YAP1, yes-associated protein 1.

Zebrafish nedd8 enhances the transcriptional activity of yap1

To assess whether zebrafish nedd8 functions similarly to mammalian NEDD8 in enhancing YAP1 transcriptional activity, we also used the 8xGTIIC promoter luciferase reporter assays for testing. Consistent with the results, nedd8 overexpression activated the yap1-driven promoter activity (Fig. 8, A and E), whereas MLN4924 treatment inhibited the yap1-driven promoter activity (Fig. 8, B and F). Furthermore, overexpression of E1 (uba3) and E2 (ubc12) also significantly enhanced the yap1-driven promoter activity (Fig. 8C). Conversely, overexpression of senp8, a potent deneddylation enzyme, was consistent with the effect of MLN4924 treatment (Fig. 8D). These data suggest that zebrafish nedd8 promotes yap1 transcriptional activity and functions similarly to mammalian NEDD8.

Figure 8.

Figure 8

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Zebrafish nedd8 enhances yap1 transcriptional activity.A, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-yap1 (200 ng) transfected HEK293T cells with or without HA-nedd8 for 36 h. B, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-yap1 (200 ng) transfected HEK293T cells with or without 1 μM MLN4924 treatment for 24 h. C, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-yap1 (200 ng) transfected HEK293T cells with or without HA-uba3 and HA-ubc12 for 36 h. D, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-yap1 (200 ng) transfected HEK293T cells with or without HA-senp8 for 36 h. E, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-yap1 (200 ng) transfected KGN cells with or without HA-nedd8 for 36 h. F, 8xGTIIC-Luc reporter activity in Myc empty vector (200 ng) or Myc-yap1 (200 ng)-transfected KGN cells with or without MLN4924 (1 μM) treatment for 24 h. Four separate experiments were performed, with each measurement performed at least three times. Data are expressed as mean ± SD. (unpaired two-tailed Student's t test). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. ns, not significant; YAP1, yes-associated protein 1.

Xiap mediates yap1 neddylation

YAP1 is evolutionarily conserved between human, rabbit, and zebrafish (Fig. S2A). To determine whether zebrafish yap1 was indeed modified by neddylation, in vitro and in vivo neddylation assays were performed. When nedd8 and yap1 were co-overexpressed in HEK293T cells, yap1 migrated with high molecular weight bands (Fig. 9A), while a faint band was observed upon coexpression of mst1 and nedd8 (Fig. S1). The off-size bands disappeared in cells expressing nedd8-ΔGG, a mutant that is unable to covalently bind to its substrates (Fig. 9B). These data suggest that the off-size bands correspond to nedd8-conjugated yap1. In addition, nedd8-conjugated yap was decreased when treated with the E1 inhibitor, MLN4924 (Fig. 9C). In contrast, overexpression of E1 (uba3) and E2 (ubc12) increased nedd8-bound yap (Fig. 9C). In vivo neddylation assay in zebrafish brain further confirmed that yap1 was neddylated (Fig. 9D). These observations suggest that yap1 is modified by neddylation.

Figure 9.

Figure 9

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Xiap may act as an E3 ligase to catalyze Yap1 neddylation.A, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with plasmids expressing HA-yap1 and PCI-His empty vector or His-nedd8 for 24 h. B, neddylation of yap1 is abolished by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with plasmids expressing Myc-yap1 and a conjugable mutant nedd8 (His-nedd8-ΔGG) for 24 h. C, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with Myc-yap1, His-nedd8, and treated with MLN4924 (1 μM) for 12 h (third lane) or transfected with Myc-yap1, His-nedd8, HA-uba3, and HA-ubc12 (fourth lane). D, protein lysates from zebrafish brains were immunoprecipitated with mouse lgG (control) or anti-YAP1 antibody, respectively. Under partially denaturing conditions, coimmunoprecipitation was detected with anti-Nedd8 antibody. E, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with HA-nedd4a, HA-xiap, HA-smurf1, and HA-mdm2 for 24 h. F, the interaction between yap1 and xiap is detected by immunoprecipitation with anti-FLAG agarose and Western blot in HEK293T cells transfected with the indicated plasmids for 24 h. G, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with HA-xiap or xiap mutant (HA-xiap-H374A) for 24 h. IB, immunoblotting; IP, immunoprecipitation; TCL, total cell lysate; YAP1, yes-associated protein 1.

We then searched for the E3 ligase that catalyzes yap neddylation. It has been defined that nedd8 and ubiquitin often share the same E3 ligase(s) (12). After overexpression of four common E3 ligases—including nedd4a, xiap, smurf1, and mdm2—which have been shown to mediate neddylation (25, 26, 27, 28), we found that only xiap increased yap1 neddylation, whereas the others had no effect (Fig. 9E). To further validate that xiap is indeed the E3 ligase that mediates yap1 neddylation, we first examined whether xiap could bind to yap1. In HEK293T cells, overexpressed HA-xiap was coimmunoprecipitated with overexpressed Flag-yap1 (Fig. 9F). However, overexpression of the catalytically inactive mutant of xiap, xiap-H374A (29), did not result in nedd8 binding to yap1 (Fig. 9G). These data suggest that xiap is a potential E3 ligase for catalyzing yap1 neddylation.

Yap1 is neddylated at lysine 159

We then sought to determine which lysine(s) in yap1 is (are) neddylated. The zebrafish yap1 protein contains 12 lysines in total (Fig. S2B). We constructed a series of mutants, in which all lysine residues were substituted with arginine residues. By in vitro neddylation assays, yap1 neddylation was decreased in the presence of K62R, K159R, and K208R/K210R mutants (Fig. 10A), suggesting that K62, K159, and K208/K210 could be the potential neddylation sites. Notably, these four sites are evolutionarily conserved (Fig. S2A). To further confirm this observation, we examined whether the cellular localization of yap1 was affected by these mutations. Interestingly, only the K159R mutation caused a decrease in yap1 protein levels in the nuclear fraction compared to the WT yap1 (Fig. 10B). Immunofluorescence staining confirmed this observation (Fig. 10C). In addition, promoter assays showed that overexpression of the K159R mutant did not enhance the transcriptional activity of YAP1 (Fig. 10D). Furthermore, an in vitro neddylation assay confirmed the neddylation of yap1 at lysine 159 (Fig. 10E). These data suggest that K159 of yap1 is a potential key neddylation site.

Figure 10.

Figure 10

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Xiap catalyzes Yap1 neddylation at lysine 159 (K159).A, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with His-nedd8 and WT Myc-yap1 or one of the site-mutated yap1 constructs for 24 h. B, protein level of yap1 in nuclei is detected by Western blot analysis in HEK293T cells transfected with Myc-yap1-WT, K62R, K159R, or K208R/K210R. α-Tubulin and H3 are used as markers for the cytoplasmic and nuclear fractions, respectively. C, fluorescence of Myc is detected by confocal microscopy in HEK293T cells transfected with Myc-yap1 or yap1 site-mutant plasmids (K62R, K159R, K208R/K210R) for 24 h. The scale bar represents 100 μm. D, relative luciferase activity of the 8xGTIIC-Luc promoter in HEK293T cells expressing the indicated constructs together with or without His-nedd8. Four separate experiments were performed, with each measurement performed at least three times. E, neddylation of yap1 is detected by immunoprecipitation with Ni-NTA agarose and Western blot in HEK293T cells transfected with Myc-yap1-WT or Myc-yap1-K159R for 24 h. Data are expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.IB, immunoblotting; IP, immunoprecipitation; TCL, total cell lysate; ns, not significant; YAP1, yes-associated protein 1.

Discussion

Neddylation has been shown to influence the activity, stability, and subcellular localization of substrate proteins (30). In this study, we demonstrate that neddylation activates Hippo-YAP signaling by enhancing the transcriptional activity and nuclear translocation of YAP1. Accumulating evidence suggests that the Hippo–YAP signaling pathway is regulated by various forms of PTMs, including phosphorylation, ubiquitination, and sumoylation, among others (31). However, whether neddylation is involved in the regulation of Hippo–YAP signaling pathway remains poorly understood. Here, we identify YAP1 is neddylated at Lys159 potentially catalyzed by XIAP, resulting in the enhancement of YAP1 transcriptional activity and nuclear translocation, revealing a novel PTM of the Hippo–YAP signaling pathway for affecting its function.

The core of the Hippo signaling pathway consists of a series of kinases (32). When the Hippo pathway is activated, a phosphorylated kinase cascade, starting with MST1/2 and progressing to LATS1/2, promotes the phosphorylation of YAP, leading to its retention in the cytoplasm for degradation. Conversely, when Hippo signaling is inhibited, YAP1 is able to enter the nucleus and carry out its transcriptional function (33, 34). During development, several studies have reported that several factors can reduce the phosphorylation of YAP1 by repressing the Hippo kinases MST1/2 and LATS1/2 (35, 36, 37). In addition, NEDD8 promotes MST1 degradation through the E3 ligase cullin 7, thereby activating YAP1 signaling for cardiomyocyte proliferation (22). Thus, the phosphorylation of upstream kinases serves as a critical regulatory mechanism that modulates YAP1 activity. Our study demonstrates that neddylation can activate the YAP1 signaling by modifying YAP1, and further analysis reveals that yap1 is conjugated by nedd8 in vivo. While inhibition of neddylation leads to the accumulation of MST1, the modification of mst1 by neddylation is comparatively weak in zebrafish. We hypothesized that neddylation regulates Hippo-YAP signaling in two distinct ways: by neddylating YAP1 to activate YAP-mediated signaling, and likely similarly, by modifying MST1 to inhibit the kinase cascade and thereby activate YAP1.

MLN4924, a potent inhibitor of the neddylation pathway, has been shown in previous studies to induce cell apoptosis and cell cycle arrest through multiple mechanisms. Specifically, MLN4924 treatment induces cell apoptosis by activating the p53 signaling pathway. In addition, MLN4924 induces the accumulation of the CRL substrate c-Myc, transactivates NOXA, and induces apoptosis (38, 39). Furthermore, we have previously shown that MLN4924 treatment in rabbit GCs caused the upregulation of proapoptotic gene expression (19). In the current study, we demonstrated that the expression of apoptosis-related genes was more significantly altered in cells cotreated with VP and MLN4924, further confirming the regulation of neddylation in YAP1 signaling. YAP1 plays a critical role in the inhibition of apoptosis. MiR-484 directly targets the mRNA of Yap1 to promote apoptosis in LPS-treated H9c2 cells (40). Lv et al. found that YAP1 knockout driven by the Foxl2 promoter resulted in increased apoptosis in GCs (24). Our results extend on these observations and show that inhibition of YAP1 signaling stimulates MLN4924-induced apoptosis of GCs.

This study also shows that the reduction of YAP1 neddylation occurs in K62R, K159R, and K208R/K210R, while only K159R reduces YAP1 nuclear localization. Therefore, K159 may be the key neddylation site controlling YAP1 localization and activity. Notably, K62R and K208R/K210R mutations show a significant decrease in the activation of the YAP/TAZ promoter activity. These lysine residues may be involved in other modifications that regulate YAP1 activity. Indeed, PTMs of YAP1 are increasingly recognized as essential contributors to ovarian development (4, 31, 41, 42). Our study demonstrates that YAP1 is modified by neddylation, verified the critical target residue for this modification, and revealed a previously unrecognized PTM of YAP1. The regulation of neddylation and the dysregulation of Hippo signaling in cancer have been extensively studied (43, 44, 45), and our findings will pave the way for new avenues in precision cancer therapy.

Experimental procedures

Rabbits, zebrafish, and cell lines

Healthy and sexually mature New Zealand white rabbits were purchased from Henan Chunying Biotechnology Co, Ltd and allowed to eat and drink freely during breeding.

Zebrafish strain AB were maintained at 28.5 °C with a photoperiod of 14 h of light and 10 h of darkness and fed regularly in a recirculating water system according to standard protocol. All experimental protocols were approved by and conducted in accordance with the Ethical Committee of Experimental Animal Care, Henan Agricultural University (Permit 2021031504).

HEK293T cells, H1299 cells, and KGN cell lines (originally obtained from American Type Culture Collection (https://www.atcc.org)) and GCs (obtained from the rabbit ovaries) (46) were grown at 37 °C in a humidified incubator containing 5% CO2. HEK293T and H1299 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Price) supplement with 10% fetal bovine serum and 1% penicillin/streptomycin (Price). KGN cells were cultured in DMEM/F12 (Price) supplement with 10% fetal bovine serum and 1% penicillin/streptomycin (Price). GCs were cultured in DMEM/F12 medium (Price), 15% fetal bovine serum (Price, China), and 1% penicillin/streptomycin (Price).

Generation of nedd8-null zebrafish

The nedd8-null zebrafish line was created through CRISPR/Cas9-mediated mutagenesis. The method for producing nedd8-null zebrafish has been previously described (17).

Plasmid construction

The rabbit YAP1 (Gene ID: 100341323) construct was generated by cloning into pCMV-Myc and mCherry-N1 vectors (Clontech Laboratories). The rabbit NEDD8 (Gene ID: 100009008) was amplified and cloned the coding sequence into pCI-his, pCMV-HA, and pcDNA3.1-EGFP vectors (Clontech Laboratories). The zebrafish yap1 (Gene ID: 561411) was amplified and cloned the coding sequence into pCMV-HA, pCMV-Myc, and pCMV-Flag (Clontech Laboratories). All zebrafish yap1 mutants (lysine-to-arginine) were generated using PCR-based mutagenesis and subcloned into the pCMV-Myc vector. Zebrafish nedd8, uba3, ubc12, and senp8 were used as described previously (17). The ORFs of zebrafish mst1 (Gene ID: 259260), nedd4a (Gene ID: 619412), smurf1 (Gene ID: 321695), mdm2 (Gene ID: 30637), xiap (ID: 373108), and the enzymatic mutants of xiap (xiap-H375A) were PCR-amplified and subcloned into pCMV-HA vectors. All constructs were confirmed by DNA sequencing. The 8xGTIIC-luciferase reporter (# 34615) was obtained from Addgene.

Antibodies and chemical reagents

Anti-NEDD8 antibody (#MAB49361) was purchased from R&D. Anti-YAP1 antibody (#A1002) was purchased from ABclonal. Antibodies including anti-Phospho-YAP1 (S127) (#GB114060), anti-MST1 (#GB11858), and anti-β-actin (#GB11001) were purchased from Servicebio. Antibodies including anti-CTGF (#WL02602), anti-H3 (WL0984a), anti-α-tubulin (#WL02296), and anti-Birc5 (#WL03492) were purchased from Wanleibio. Anti-CDKN1A antibody (#2947T) and normal rabbit IgG (#2729) were purchased by Cell Signaling Technology. Anti-Myc (#sc-40) antibody was purchased from Santa Cruz (Santa Cruz). Anti-HA antibody (#901515) was purchased from Covance. Anti-GAPDH antibody was purchased from Proteintech. Anti-Flag antibody (#F1804) and guanidine hydrochloride (#WXBD4283 V) were purchased from Sigma. Protein G Sepharose (#17-0618-01) was purchased from GE HealthCare Company. Ni-NTA Agarose (#169045429) was purchased from Gingen. Imidazole (#10429010130) was purchased from Genview. MLN-4924 (MCE) was dissolved in dimethyl sulfoxide and added into the cell culture medium at a final concentration of 1 μM as previously described (19). VP (MCE) was dissolved in dimethyl sulfoxide and added to the cell culture medium at the desired concentration.

Immunoprecipitation and Western blot analysis

HEK293T cells were transfected with different combinations for 24 h, and then the cells were lysed in radio immunoprecipitation assay lysis buffer containing 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA (pH 8.0), 150 mM NaCl, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, and a 1:100 dilution of protease inhibitor mixture (Sigma-Aldrich), After incubation for 30 min at 4 °C, lysates were collected and centrifuged at 10,000g at 4 °C for 15 min, protein concentrations were measured, and equal amounts of lysates were used for coimmunoprecipitation (co-IP). Co-IP was performed with the indicated beads and antibodies. The precipitants were then washed three times with radio immunoprecipitation assay buffer, and the immunocomplexes were boiled with 1× SDS sample loading buffer, separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). Western blot was performed as described previously (47). The QuickChemi 5200 Chemiluminescence Imaging system (GD50202) was used to photograph the blots. The band intensity was quantified using Image J software (https://imagej.nih.gov/ij/).

In vitro neddylation and ubiquitylation assays

HEK293T cells were transfected with the indicated constructs for 16 to 22 h, and then the cells were harvested. For MLN4924 treatments, after the cells were transfected for 12 h, MLN4924 was added into the medium and incubated for 12 h, and then the cells were harvested. The cells were lysed using the lysis buffer (6 M guanidinium-HCL, 0.1 M Na2HPO4/NaH2PO4 (pH = 8.0), 10 mM Imidazole). After ultrasonic crushing, the lysates were incubated with prewashed Ni-NTA agarose beads (Qiagen) in lysis buffer and rotated at 4 °C overnight. The beads were washed three times with washing buffer I (one-fifth lysis buffer plus four-fifths washing buffer II) and three times with washing buffer II (25 mM Tris–HCL (pH = 6.8), 20 mM imidazole). Subsequently, the beads were eluted with the sample-loading buffer and immunoblotting with the indicated antibodies.

In vivo neddylation

Zebrafish brains dissected from nearly 20 males were lysed with modified lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1% SDS, 1 mM Na3VO4, 1 mM DTT, and 10 mM NaF] supplemented with a protease inhibitor cocktail. After incubation at 100 °C for 10 min, the lysate was diluted ten times with modified lysis buffer without SDS. The lysates were then incubated with the indicated antibody for 3 h at 4 °C. Protein A/G-plus agarose beads (Santa Cruz Biotechnology) were added, and the lysates were rotated gently for 8 h at 4 °C. The immunoprecipitates were washed at least three times in wash buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1 mM DTT, and 10 mM NaF]. Proteins were recovered by boiling the beads in 2× SDS sample buffer and analyzed by Western blot. Immunoblotting and Co-IP were performed using the indicated antibodies.

Luciferase reporter assay

HEK293T cells or KGN cells were seeded in 24-well plates and cotransfected with the indicated luciferase reporter plasmids and expression vectors by VigoFect (Vigorous Biotech), together with pCMV-Renilla as an internal control. After transfection 36 h, the cells were harvested for luciferase assays. The luciferase activity was determined by the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Firefly luciferase activity was normalized with Renilla luciferase activity. For MLN4924 treatment, after the cells were transfected for 12 h, MLN4924 was added to the culture medium and incubated for 24 h, and then the cells were collected for analysis. Data are reported as means ± SD of three repeated experiments (n = 3).

Fluorescence microscopy

H1299 cells were transfected with rabbit EGFP-NEDD8 and mCherry-YAP1 vectors. At 24 h posttransfection, the cells were washed twice with PBS and then were stained with 4′, 6-diamidino-2-phenylindole (Beyotime) (1 μg/ml) for 15 min in the dark at room temperature. Finally, the coverslips were washed and imaged with a confocal microscope under a 63× oil immersion objective (SP8; Leica Microsystems).

Immunofluorescence and confocal microscopy

Immunofluorescence staining for rabbit ovaries and cells was conducted as previously described (48, 49). The antibodies used for immunofluorescence are mentioned above.

Quantitive real-time PCR

Total RNA was extracted from tissues and cells using the TRIzol reagent (TransGen Biotech) following the recommended protocol. Complementary DNA was synthesized using HiScript II first Strand cDNA Synthesis Kit (Vazyme). Quantitive real-time PCR assays were conducted using MonAmp SYBR Green qPCR Mix (Monad Bio). Primers are listed in Table S1.

RNA-Seq and analysis

Total RNAs were extracted using TRIzol reagent (Invitrogen), and the quality and concentration were assessed by a NanoDrop spectrophotometer (Thermo Fisher Scientific). More than 1 μg of total RNA was used to prepare the total RNA-Seq libraries using NEB Next UItra Directional RNA Library Prep Kit. Libraries were accessed using Agilent 2100 Bioanalyzer (Agilent, 2100), Agilent High Sensitivity DNA Kit (Agilent, 5067-4626) and detected concentration by Pico green (Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, P7589). Libraries were sequenced using the NovaSeq 6000 platform (Illumina) with PE150 mode. Reads were aligned to the rabbit reference genome. The gene expression data was analyzed using HTSeq for statistical analysis of read counts, and fragments per kilobase million was employed to normalize gene expression levels. DESeq was then used to analyze differential gene expression, with screening conditions set as follows: expression difference multiple |log2 FoldChange|>1.2, significant p value <0.05. Additionally, the pheatmap package in R was utilized to perform two-way clustering analysis, generating a heat map based on expression levels and patterns. Furthermore, gene ontology enrichment and Kyoto encyclopedia of genes and genomes analysis of DEGs were conducted on the Personal Cloud Platform (https://www.genescloud.cn/) (50, 51, 52).

Statistical analysis

Data were analyzed using SPSS 26.0 software (https://www.ibm.com/cn-zh/spss) and GraphPad Prism v8.0.2 software (https://www.graphpad-prism.cn/) for mapping. Other experiments were performed using unpaired two-tailed Student’s t test or two-way ANOVA analysis. All data were presented as means ± SD. A value of p < 0.05 was deemed to be statistically significant. Data are representative of three independent experiments performed in three technical repeats.

Data availability

Further information and requests for resources and reagents should be directed to and will be fulfilled by G. Y.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgments

We are grateful to Fang Zhou (Institute of Hydrobiology, Chinese Academy of Sciences) for her help with fluorescent microscope analysis.

Author contributions

M. C., Y. L., W. X., and G. Y. conceptualization; M. C. data curation; M. C., M. Z., C. G., Y. D., H. X., B. L., and M. L. investigation; M. C. and Y. L. methodology; M. C., M. Z., C. G., Y. D., H. X., B. L., and M. L. visualization; M. C., W. X., and G. Y. writing–original draft; W. X. and G. Y. supervision; W. X. and G. Y. writing–review and editing; G. Y. validation.

Funding and additional information

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24010308 to W.X.); National Natural Science Foundation of China (32102833 to G.Y.); China Postdoctoral Science Foundation (2021M701107 to G.Y.); and State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences (2021FB08 to G.Y.). Supplementary information is available at Journal of Biological Chemistry’s website.

Reviewed by members of the JBC Editorial Board. Edited by Brian Strahl

Contributor Information

Wuhan Xiao, Email: w-xiao@ihb.ac.cn.

Guangqing Yu, Email: ygq@henau.edu.cn.

Supporting information

Supporting Information
mmc1.docx (284.1KB, docx)

References

  • 1.Yu F.X., Zhao B., Guan K.L. Hippo pathway in organ size control, tissue Homeostasis, and cancer. Cell. 2015;163:811–828. doi: 10.1016/j.cell.2015.10.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kawamura K., Cheng Y., Suzuki N., Deguchi M., Sato Y., Takae S., et al. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proc. Natl. Acad. Sci. U. S. A. 2013;110:17474–17479. doi: 10.1073/pnas.1312830110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hu L.L., Su T., Luo R.C., Zheng Y.H., Huang J., Zhong Z.S., et al. Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice. J. Cell. Physiol. 2019;234:1578–1587. doi: 10.1002/jcp.27024. [DOI] [PubMed] [Google Scholar]
  • 4.Plewes M.R., Hou X., Zhang P., Liang A., Hua G., Wood J.R., et al. Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro. Biol. Reprod. 2019;101:1001–1017. doi: 10.1093/biolre/ioz139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Dong J., Feldmann G., Huang J., Wu S., Zhang N., Comerford S.A., et al. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell. 2007;130:1120–1133. doi: 10.1016/j.cell.2007.07.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Wang L., Choi K., Su T., Li B., Wu X., Zhang R., et al. Multiphase coalescence mediates Hippo pathway activation. Cell. 2022;185:4376–4393. doi: 10.1016/j.cell.2022.09.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Oh H., Irvine K.D. In vivo regulation of Yorkie phosphorylation and localization. Development. 2008;135:1081–1088. doi: 10.1242/dev.015255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Halder G., Johnson R.L. Hippo signaling: growth control and beyond. Development. 2011;138:9–22. doi: 10.1242/dev.045500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kerscher O., Felberbaum R., Hochstrasser M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 2006;22:159–180. doi: 10.1146/annurev.cellbio.22.010605.093503. [DOI] [PubMed] [Google Scholar]
  • 10.Zhou Q., Zheng Y., Sun Y. Neddylation regulation of mitochondrial structure and functions. Cell Biosci. 2021;11:55. doi: 10.1186/s13578-021-00569-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Pan Z.Q., Kentsis A., Dias D.C., Yamoah K., Wu K. Nedd8 on cullin: building an expressway to protein destruction. Oncogene. 2004;23:1985–1997. doi: 10.1038/sj.onc.1207414. [DOI] [PubMed] [Google Scholar]
  • 12.Enchev R.I., Schulman B.A., Peter M. Protein neddylation: beyond cullin-RING ligases. Nat. Rev. Mol. Cell Biol. 2015;16:30–44. doi: 10.1038/nrm3919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang L., Jing H., Li H., Chen W., Luo B., Zhang H., et al. Neddylation is critical to cortical development by regulating Wnt/β-catenin signaling. Proc. Natl. Acad. Sci. U. S. A. 2020;117:26448–26459. doi: 10.1073/pnas.2005395117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Vogl A.M., Brockmann M.M., Giusti S.A., Maccarrone G., Vercelli C.A., Bauder C.A., et al. Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition. Nat. Neurosci. 2015;18:239–251. doi: 10.1038/nn.3912. [DOI] [PubMed] [Google Scholar]
  • 15.Soucy T.A., Smith P.G., Milhollen M.A., Berger A.J., Gavin J.M., Adhikari S., et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458:732–U767. doi: 10.1038/nature07884. [DOI] [PubMed] [Google Scholar]
  • 16.Wang B., Wang T., Zhu H., Yan R., Li X., Zhang C., et al. Neddylation is essential for β-catenin degradation in Wnt signaling pathway. Cell Rep. 2022;38 doi: 10.1016/j.celrep.2022.110538. [DOI] [PubMed] [Google Scholar]
  • 17.Yu G., Liu X., Zhang D., Wang J., Ouyang G., Chen Z., et al. Zebrafish Nedd8 facilitates ovarian development and the maintenance of female secondary sexual characteristics via suppression of androgen receptor activity. Development. 2020;147 doi: 10.1242/dev.194886. [DOI] [PubMed] [Google Scholar]
  • 18.Kostrhon S., Prabu J.R., Baek K., Horn-Ghetko D., von Gronau S., Kluegel M., et al. CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation. Nat. Chem. Biol. 2021;17:1075–1083. doi: 10.1038/s41589-021-00858-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Chen M., Liu Y., Zuo M., Zhang M., Wang Z., Li X., et al. Integrated analysis reveals the regulatory mechanism of the neddylation inhibitor MLN4924 on the metabolic dysregulation in rabbit granulosa cells. BMC Genomics. 2024;25:254. doi: 10.1186/s12864-024-10118-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zhang Z., Du J., Wang S., Shao L., Jin K., Li F., et al. OTUB2 promotes cancer Metastasis via Hippo-independent activation of YAP and TAZ. Mol. Cell. 2019;73:7. doi: 10.1016/j.molcel.2018.10.030. [DOI] [PubMed] [Google Scholar]
  • 21.Yan C., Yang H., Su P., Li X., Li Z., Wang D., et al. OTUB1 suppresses Hippo signaling via modulating YAP protein in gastric cancer. Oncogene. 2022;41:5186–5198. doi: 10.1038/s41388-022-02507-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zou J., Ma W., Li J., Littlejohn R., Zhou H., Kim I.-m., et al. Neddylation mediates ventricular chamber maturation through repression of Hippo signaling. Proc. Natl. Acad. Sci. U. S. A. 2018;115:E4101–E4110. doi: 10.1073/pnas.1719309115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Harvey K.F., Zhang X., Thomas D.M. The Hippo pathway and human cancer. Nat. Rev. Cancer. 2013;13:246–257. doi: 10.1038/nrc3458. [DOI] [PubMed] [Google Scholar]
  • 24.Li H., Wang X., Mu H., Mei Q., Liu Y., Min Z., et al. Mir-484 contributes to diminished ovarian reserve by regulating granulosa cell function via YAP1-mediated mitochondrial function and apoptosis (vol 18, pg 1008, 2022) Int. J. Biol. Sci. 2023;19:3288–3289. doi: 10.7150/ijbs.84828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Du M.G., Liu F., Chang Y., Tong S., Liu W., Chen Y.-j., et al. Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis. J. Biol. Chem. 2021;297 doi: 10.1016/j.jbc.2021.101307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Nagano T., Hashimoto T., Nakashima A., Kikkawa U., Kamada S. X-linked inhibitor of apoptosis protein mediates neddylation by itself but does not function as a NEDD8-E3 ligase for caspase-7. FEBS Lett. 2012;586:1612–1616. doi: 10.1016/j.febslet.2012.04.056. [DOI] [PubMed] [Google Scholar]
  • 27.Xirodimas D.P., Saville M.K., Bourdon J.C., Hay R.T., Lane D.P. Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity. Cell. 2004;118:83–97. doi: 10.1016/j.cell.2004.06.016. [DOI] [PubMed] [Google Scholar]
  • 28.Tu J., Zhang B., Fang G., Chang W., Zhao Y. Neddylation-mediated Nedd4-2 activation regulates ubiquitination modification of renal NBCe1. Exp. Cell Res. 2020;390 doi: 10.1016/j.yexcr.2020.111958. [DOI] [PubMed] [Google Scholar]
  • 29.Xie P., Peng Z., Chen Y., Li H., Du M., Tan Y., et al. Neddylation of PTEN regulates its nuclear import and promotes tumor development. Cell Res. 2021;31:291–311. doi: 10.1038/s41422-020-00443-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Watson I.R., Irwin M.S., Ohh M. NEDD8 pathways in cancer, sine Quibus Non. Cancer Cell. 2011;19:168–176. doi: 10.1016/j.ccr.2011.01.002. [DOI] [PubMed] [Google Scholar]
  • 31.He M., Zhou Z., Shah A.A., Hong Y., Chen Q., Wan Y. New insights into posttranslational modifications of Hippo pathway in carcinogenesis and therapeutics. Cell Div. 2016;11:1–10. doi: 10.1186/s13008-016-0013-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Thines L., Gorisse L., Li Z., Sayedyahossein S., Sacks D.B. Calmodulin activates the Hippo signaling pathway by promoting LATS1 kinase-mediated inhibitory phosphorylation of the transcriptional coactivator YAP. J. Biol. Chem. 2022;298 doi: 10.1016/j.jbc.2022.101839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yu F.X., Guan K.L. The Hippo pathway: regulators and regulations. Genes Dev. 2013;27:355–371. doi: 10.1101/gad.210773.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Xu Y., Qu M., He Y., He Q., Shen T., Luo J., et al. Smurf1 polyubiquitinates on K285/K282 of the kinases Mst1/2 to attenuate their tumor-suppressor functions. J. Biol. Chem. 2023;299:105395. doi: 10.1016/j.jbc.2023.105395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Saito A., Nagase T. Hippo and TGF-β interplay in the lung field. Am. J. Physiol. Lung Cell. Mol. Physiol. 2015;309:L756–L767. doi: 10.1152/ajplung.00238.2015. [DOI] [PubMed] [Google Scholar]
  • 36.Wu N., Quy N., Wan Y., Zhou T., Venter J., Frampton G.A., et al. The Hippo signaling functions through the Notch signaling to regulate intrahepatic bile duct development in mammals. Lab. Invest. 2017;97:843–853. doi: 10.1038/labinvest.2017.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Heallen T., Zhang M., Wang J., Bonilla-Claudio M., Klysik E., Johnson R.L., et al. Hippo pathway inhibits Wnt signaling to Restrain cardiomyocyte proliferation and heart size. Science. 2011;332:458–461. doi: 10.1126/science.1199010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Chen Y., Sun L. Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway. Biosci. Rep. 2022;42 doi: 10.1042/BSR20220994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Knorr K.L.B., Schneider P.A., Meng X.W., Dai H., Smith B.D., Hess A.D., et al. MLN4924 induces Noxa upregulation in acute myelogenous leukemia and synergizes with Bcl-2 inhibitors. Cell Death Differ. 2015;22:2133–2142. doi: 10.1038/cdd.2015.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Xu M., Li X.-Y., Song L., Tao C., Fang J., Tao L. miR-484 targeting of Yap1-induced LPS-inhibited proliferation, and promoted apoptosis and inflammation in cardiomyocyte. Biosci. Biotechnol. Biochem. 2021;85:378–385. doi: 10.1093/bbb/zbaa009. [DOI] [PubMed] [Google Scholar]
  • 41.Clark K.L., Davis J.S. Perfluorooctanoic acid (PFOA) promotes follicular growth and alters expression of genes that regulate the cell cycle and the Hippo pathway in cultured neonatal mouse ovaries. Toxicol. Appl. Pharmacol. 2022;454 doi: 10.1016/j.taap.2022.116253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Lin K.C., Park H.W., Guan K.L. Regulation of the Hippo pathway transcription factor TEAD. Trends Biochem. Sci. 2017;42:862–872. doi: 10.1016/j.tibs.2017.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Jia X., Li C., Li L., Liu X., Zhou L., Zhang W., et al. Neddylation Inactivation facilitates FOXO3a nuclear Export to suppress Estrogen receptor transcription and Improve Fulvestrant Sensitivity. Clin. Cancer Res. 2019;25:3658–3672. doi: 10.1158/1078-0432.CCR-18-2434. [DOI] [PubMed] [Google Scholar]
  • 44.Calses P.C., Crawford J.J., Lill J.R., Dey A. Hippo pathway in cancer: aberrant regulation and therapeutic opportunities. Trends Cancer. 2019;5:297–307. doi: 10.1016/j.trecan.2019.04.001. [DOI] [PubMed] [Google Scholar]
  • 45.Abidi N., Xirodimas D.P. Regulation of cancer-related pathways by protein NEDDylation and strategies for the use of NEDD8 inhibitors in the clinic. Endocr. Relat. Cancer. 2015;22:T55–T70. doi: 10.1530/ERC-14-0315. [DOI] [PubMed] [Google Scholar]
  • 46.Song G., Jiang Y., Wang Y., Song M., Niu X., Xu H., et al. Modulation of Cathepsin S CTSS regulates the Secretion of Progesterone and Estradiol, proliferation, and apoptosis of ovarian granulosa cells in rabbits. Animals. 2021;11:1770. doi: 10.3390/ani11061770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yu G., Li X., Zhou Z., Tang J., Wang J., Liu X., et al. Zebrafish phd3 negatively regulates Antiviral responses via suppression of Irf7 transactivity independent of its Prolyl Hydroxylase activity. J. Immunol. 2020;205:1135–1146. doi: 10.4049/jimmunol.1900902. [DOI] [PubMed] [Google Scholar]
  • 48.Wang Z., Chen X., Fan S., Zhu C., Deng H., Tang J., et al. Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity. J. Biol. Chem. 2022;298 doi: 10.1016/j.jbc.2022.102633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Zhu J., Zhang D., Liu X., Yu G., Cai X., Xu C., et al. Zebrafish prmt5 arginine methyltransferase is essential for germ cell development. Development. 2019;146 doi: 10.1242/dev.179572. [DOI] [PubMed] [Google Scholar]
  • 50.Ogata H., Goto S., Sato K., Fujibuchi W., Bono H., Kanehisa M. KEGG: Kyoto Encyclopedia of genes and genomes. Nucleic Acids Res. 1999;27:29–34. doi: 10.1093/nar/27.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Kanehisa M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 2019;28:1947–1951. doi: 10.1002/pro.3715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Kanehisa M., Furumichi M., Sato Y., Kawashima M., Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023;51:D587–D592. doi: 10.1093/nar/gkac963. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Supporting Information
mmc1.docx (284.1KB, docx)

Data Availability Statement

Further information and requests for resources and reagents should be directed to and will be fulfilled by G. Y.

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