RNF114 is a newly discovered partner of PARP10
Recently, we identified Aurora A as a functional partner of PARP10 through tandem affinity purification. In addition to Aurora A, RNF114, a ubiquitin E3 ligase, was found in the PARP10 complex, suggesting that RNF114 might also be a PARP10-interacting protein. Then, we performed exogenous and endogenous reciprocal immunoprecipitation (IP) assays to verify the interaction between PARP10 and RNF114. As expected, exogenously expressed HA-RNF114 interacted with SFB-PARP10, and vice versa (Figure 1a and 1b). Endogenous PARP10 also interacted with RNF114, as determined by a co-IP assay using an anti-PARP10 antibody (Figure 1c). Taken together, these results indicate that RNF114 is a previously unknown partner of PARP10.
Auto-mono-ADP-ribosylation of PARP10 is required for its association with RNF114
To gain further understanding of the interaction between PARP10 and RNF114, we generated serial deletion mutants of PARP10 to identify the region of PARP10 that is essential for its association with RNF114. As shown in Figure 2a, deletion of the 700-1025 aa region of PARP10, which contains the catalytic domain, abolished the interaction between PARP10 and RNF114, suggesting that the catalytic domain of PARP10 is critical for its association with RNF114. Then, we generated an enzymatically inactive PARP10 mutant to investigate whether the interaction between PARP10 and RNF114 is dependent on the enzymatic activity of PARP10. As shown in Figure 2b, the G888W mutant, which abolished the enzymatic activity of PARP10, did interact with RNF114, as indicated by the co-IP assay results. Interestingly, we found that His-PARP10, which was purified from E. coli, did not interact with SFB-RNF114, which is expressed in 293T cells. However, after auto-mono-ADP-ribosylation in vitro, the mono-ADP-ribosylated His-PARP10 efficiently pulled down SFB-RNF114, suggesting that the auto-mono-ADP-ribosylation of PARP10 is required for its association with RNF114 (Figure 2c). Taken together, these results indicate that the interaction between RNF114 and PARP10 is dependent on the auto-mono-ADP-ribosylation of PARP10.
RNF114 ubiquitinates PARP10 through K27-linked ubiquitination
Both PARP10 and RNF114 are enzymes, raising the possibility that RNF114 is a substrate of PARP10 or PARP10 is a substrate of RNF114. To identify the link between modified PARP10 and RNF114, we first performed in vitro mono-ADP-ribosylation assays using biotin-NAD+ as a donor to examine whether PARP10 can modify RNF114 via mono-ADP-ribosylation. As shown in Supplementary Figure 1, PARP10 was modified by auto-mono-ADP-ribosylation, as shown by western blotting with an anti-biotin antibody. However, RNF114 was not modified by PARP10 mono-ADP-ribosylation, as shown by in vitro assay, suggesting that RNF114 may not be a PARP10 substrate in vitro.
Therefore, we performed ubiquitination assays to determine whether PARP10 is an RNF114 substrate. As shown in Figure 3a, co-expression of GFP-RNF114 with Flag-PARP10 dramatically increased the ubiquitination level of Flag-PARP10 in 293T cells. Compared with wild-type RNF114, the RNF114 C29G/C32G mutant, which abolishes the E3 ligase activity of RNF114, did not promote the ubiquitination of PARP10 (Figure 3b), suggesting that RNF114 mediates the ubiquitination of PARP10 in a manner dependent on the RNF114 E3 ligase activity.
To investigate the ubiquitination of PARP10 by RNF114, we generated RNF114-knockout HeLa cells by CRISPR-Cas9 editing. We obtained two individual RNF114-knockout cell lines. The RNF114-knockout cells, which expressed no RNF114 protein, was examined by western blotting using anti-RNF114 antibody (Supplementary Figure 2a) and was found to contain several base pair deletions in genomic DNA, which caused a frame-shift mutation in RNF114, as indicated by DNA sequencing (Supplementary Figure 2b).
Then, we used the RNF114-knockout HeLa cells to examine the effect of RNF114 deficiency on PARP10 ubiquitination. As shown in Figure 3c, the ubiquitination level of PARP10 was dramatically decreased in the RNF114-deficient cells compared with the level in the wild-type cells, further suggesting that PARP10 is an RNF114 substrate. However, the total level of PARP10 protein was not obviously changed in the RNF114-deficient cells compared with the wild-type cells (Figure 3c), suggesting that the ubiquitination of PARP10 by RNF114 may not promote its degradation. Next, an in vivo ubiquitination assay was performed by using a panel of ubiquitin mutants with single K/R mutations at the sites of the seven lysine residues in ubiquitin. As shown in Figure 3d, compared with wild-type and other lysine mutants, the ubiquitin K27R mutant abolished the ubiquitination of PARP10 that is mediated by RNF114. We also used the K27-only (K27O), K33-only (K33O), K48-only(K48O) and K63-only(K63O) mutants of ubiquitin to perform an in vivo ubiquitination assay. As shown in Figure 3e, the K33O, K48O and K63O mutants abolished the ubiquitination of PARP10 mediated by RNF114, while the K27O mutant had no effect on the ubiquitination of PARP10, compared with the effect of wild-type ubiquitin. Taken together, these results indicate that RNF114 ubiquitinates PARP10 through K27-linked ubiquitination.
RNF114 regulates PARP10 enzymatic activity
Emerging evidence suggests that K27-linked polyubiquitination plays important roles in the regulation of substrate function but not stability. Therefore, we examined whether RNF114-mediated ubiquitination of PARP10 affected its function. First, we examined the mono-ADP-ribosylation levels of PARP10 and its substrate Aurora A in wild-type and RNF114-knockout cells by pull-down assay using the GST-Macro domain 2 of PARP14, which specifically associates with mono-ADP-ribosylated proteins [24, 25]. As shown in Figure 4a and 4b, PARP10 and Aurora A were pulled down by the GST-Macro2 domain from wild-type cells more efficiently than by the domain from the RNF114-knockout cells, suggesting that the mono-ADP-ribosylation levels of PARP10 and Aurora A were decreased after RNF114 depletion. Next, we examined the phosphorylation levels of Aurora A and its substrate Akt in wild-type and RNF114-knockout cells. As shown in Figure 4c, compared with those in the wild-type cells, the phosphorylation levels of Aurora A and Akt were increased in the RNF114-knockout cells, suggesting that RNF114 deficiency increased Aurora A kinase activity. Taken together, these results indicated that RNF114-mediated ubiquitination of PARP10 positively regulated PARP10 activity, which in turn suppressed Aurora A activity and downstream signalling.
RNF114 regulates the EMT process and tumour metastasis
Recently, we demonstrated that PARP10 is a tumour metastasis suppressor. RNF114 regulates PARP10 activity, raising the possibility that RNF114 may also play important roles in cancer cell migration and tumour metastasis. To test this hypothesis, we performed cell migration and invasion assays using wild-type and RNF114-deficient HeLa cells. As shown in Figure 5a and 5b, compared with these processes in wild-type cells, the extent of the migration and invasion of RNF114-deficient cells was dramatically increased, suggesting that RNF114 negatively regulates the migration and invasion of HeLa cells. Then, we performed an analysis of the function of RNF114 in the distant metastasis of HeLa cells injected into the tail veins of mice. Wild-type HeLa cells and two RNF114-deficient cell lines were injected into BALB/c nude mice. Eight weeks after the injection, the mice were sacrificed, and metastatic lung tumours were analysed and properties calculated. As shown in Figure 5c and 5d, compared with the tumours established by the wild-type HeLa cells, the number and size of the lung tumours derived from the RNF114-deficient HeLa cells were significantly increased, suggesting that RNF114 deficiency promoted tumour metastasis in vivo.
Since the epithelial-mesenchymal transition (EMT) is implicated in cell migration and invasion and because PARP10 is also involved in EMT regulation, the expression levels of EMT-associated markers were also analysed in the wild-type and RNF114-knockout cells. As shown in Figure 5e, compared with that in the wild-type cells, the findings showed that the expression level of the epithelial marker E-cadherin was reduced and that the expression levels of the mesenchymal markers N-cadherin and Vimentin were increased in the RNF114-deficient cells, suggesting that RNF114 deficiency promotes the EMT process.
Next, we re-expressed RNF114 in the RNF114-deficient HeLa cells to validate the function of RNF114 in the EMT process and cell migration. As shown in Figure 5f, the expression levels of wild-type RNF114 and the RNF114 mutant introduced to the RNF114-deficient HeLa cells were similar to those of endogenous RNF114 in the control cells. The re-expression of wild-type RNF114 decreased the expression levels of N-cadherin and Vimentin and increased the expression level of E-cadherin, while re-expression of the enzymatic RNF114 mutant did not lead to these outcomes (Figure 5f). Consistently, the re-expression of wild-type RNF114, but not the enzymatic RNF114 mutant, dramatically suppressed the migration of RNF114-deficient HeLa cells (Figure 5g), suggesting that RNF114 regulates the EMT process and that cancer cell migration is dependent on its ubiquitin ligase activity. Taken together, these results suggest that RNF114 is a tumour suppressor and plays important roles in the EMT process and tumour metastasis.
In addition, PARP10 is overexpressed in RNF114-deficient cells, and the migration and invasion of wild-type, RNF114-deficient and PARP10-overexpressing RNF114-deficient cells were examined to confirm that RNF114 regulates the migration and invasion of HeLa cells in a PARP10-dependent manner. As shown in Supplementary Figure 3, PARP10 overexpression inhibited the migration and invasion of RNF114-deficient cells to the same level as the wild-type cells, indicating that RNF114 mediates cell migration and invasion through the regulation of PARP10.