Interaction between HECT domain E3 ligase, Wwp2, and the large subunit of RNA polymerase II, Rpb1.
To identify novel substrates of Wwp2, we performed affinity chromatography using bacterially expressed GST-WW-HECT (WW and HECT domains of Wwp2) fusion protein and lysate from murine F9 embryonal carcinoma cells. As shown in Fig. , one band of 220 kDa was present only in the F9 lysate-containing GST-WW-HECT column and was excised for mass spectrometric analysis, which indicated that the band contained the large subunit of RNA polymerase II, Rpb1.
FIG. 1. Identification and characterization of the interaction between Wwp2 and Rpb1. (A) Coomassie blue staining of an SDS-polyacrylamide gel electrophoresis gel containing the proteins from the indicated columns is shown. (B) GST pull-down assay to show that (more ...)
To confirm the interaction between Wwp2 and Rpb1, a GST pull-down assay was performed with bacterially expressed GST-Rpb1 fusion protein and His-Wwp2 fusion protein. His-Wwp2CA fusion protein that contains a mutation at residue 838 of cysteine to alanine in the HECT domain of Wwp2 was also included in the assay. We have previously shown that cysteine residue 838 in the HECT domain of Wwp2 is essential for its enzymatic activity (42
). Western blot analysis with anti-His antibody indicated that immobilized GST-Rpb1, but not GST alone, was able to pull down His-Wwp2. In addition, the mutation at residue 838 from cysteine to alanine did not affect its association with Rpb1 (Fig. ). Theses results indicate that Wwp2 interacts with Rpb1 directly in vitro.
Next, we determined whether Wwp2 and Rpb1 interact in vivo. Coimmunoprecipitation experiments were performed with lysates from HEK 293 cells expressing Flag-Wwp2 or Flag-Wwp2CA and EGFP-CTD or EGFP. The CTD of Rpb1 was used in this experiment because it was easier to express than full-length Rpb1. As shown in Fig. , EGFP-CTD coimmunoprecipitated with Flag-Wwp2 (lane 1, row 1). Mutation of cysteine to alanine at residue 838 of the HECT domain of Wwp2 had no effect on the association of Wwp2 with EGFP-CTD in vivo (lane 5, row 1). As a negative control, EGFP alone did not coimmunoprecipitate with Flag-Wwp2 or Flag-Wwp2CA, suggesting that the interaction between EGFP-CTD and Flag-Wwp2 or Flag-Wwp2CA was mediated specifically by CTD (row 3).
To prove that endogenous Rpb1 specifically coimmunoprecipitates with endogenous Wwp2, we derived stable F9 cell lines that expressed Tc-inducible small interference RNAs (siRNA) specifically targeting Wwp2 coding sequence. The specificity of the siRNA was confirmed by Western blotting, which showed that addition of Tc to the cell culture media dramatically reduced expression of Wwp2 in the cells (Fig. , lane 2 in row 3), whereas the expression level of tubulin was not affected. Next, proteins from F9 cell lysate were immunoprecipitated with anti-Wwp2 antibody or anti-GST antibody, and precipitated proteins were analyzed by using antibody H14, or antibody C21, or anti-Wwp2 antibody and anti-tubulin antibody (Fig. ). Interestingly, both hyperphosphorylated and hypophosphorylated Rpb1 were readily detected in Wwp2-antibody-immunoprecipitated complexes without Tc treatment, although it seems that Wwp2 favored to form complex with hypophosphorylated Rpb1 (compare row 1 and row 2 of lane 4 in Fig. ). As a negative control, none of the used antibody detected signal in GST-antibody precipitated proteins (lane 3). Moreover, the fact that tubulin antibody did not produce any signal in Wwp2 antibody-immunoprecipitated proteins proves the specificity of our Wwp2 antibody (row 4 of lane 4 and 5). Compellingly, treatment of cells with Tc significantly compromised the association between Wwp2 and Rpb1, especially hypophosphorylated Rpb1. These observations clearly confirm that Wwp2 and Rpb1 specifically form a stable complex in living cells under physiological conditions. Although H14 antibody is claimed to recognize hyperphosphorylated CTD of Rpb1, they may weakly recognize hypophosphorylated CTD as well. To exclude this possibility, F9 cell lysate was treated with λ phosphatase, and the expression levels of hypophosphorylated and hyperphosphorylated Rpb1 were determined by Western blotting. As expected, treatment of cell lysate with the phosphatase completely abolished H14 antibody-recognized signal, although C21-, Wwp2-, and tubulin-antibody-recognized signals were not changed, suggesting that the Western blot band detected by H14 antibody in our experimental conditions is indeed hyperphosphorylated Rpb1 (Fig. , lanes 1 and 2). Similarly, H14 antibody could not detect any signal in Wwp2 antibody-immunoprecipitated proteins after treatment with the phosphatase (row 1 of lane 5), suggesting that the signal recognized by H14 antibody in Wwp2 antibody-immunoprecipitated proteins was indeed from hyperphosphorylated Rpb1 (row 1 of lane 4). In contrast, λ phosphatase treatment did not affect the association of hypophosphorylated Rpb1 with Wwp2. Taken together, our results demonstrate that Wwp2 specifically binds both hyper- and hypophosphorylated Rpb1 in the cells. However, the phosphorylation state of Rpb1 might affect its association with Wwp2, with the hypophosphorylated state of Rpb1 favoring the complex formation.
Mapping the interaction domains in Wwp2 and Rpb1.
GST pull-down assays were performed to identify the domains mediating the interaction between Wwp2 and Rpb1. His-Wwp2 fusion protein and the GST-CTD of Rpb1 fusion protein were expressed in bacteria and purified (Fig. , bottom panel). Western blot analysis showed that GST-CTD, but not GST alone, was capable of binding His-Wwp2 in vitro, suggesting that the CTD domain is sufficient to mediate the interaction between Wwp2 and Rpb1 (Fig. , top panel). On the other hand, WW domain (including four WW domains) of Wwp2 was found to interact with Rpb1 (Fig. ). In contrast, the HECT and C2 domains of Wwp2 did not bind Rpb1.
FIG. 2. Interacting regions between Wwp2 and Rpb1. (A) The ability of the Rpb1 CTD binds to Wwp2 was analyzed by GST pull-down assay (top panel). Bacterially expressed fusion proteins were stained with Coomassie blue (bottom panel). (B) The ability of various (more ...)
In addition, four WW domains of Wwp2 were individually expressed and purified for GST pull-down experiments. The results showed that the second WW domain was the major domain for mediating the association between Wwp2 and Rpb1 in vitro (Fig. ). Furthermore, to test whether the negative results obtained from the GST-WW1, -WW3, and -WW4 domains arose from improper folding of these isolated domains, their interaction with Srg3, a known substrate of Wwp2 (unpublished data), was examined. As shown in Fig. , GST-WW3 domain could bind Srg3 with a similar efficiency to that of GST-WW2 domain. The observation indicates that although WW domains have sequence similarity and associate with proline-rich proteins, each domain has a different specificity or affinity for protein interactions.
Wwp2 ubiquitinates Rpb1 both in vitro and in vivo.
Our group has previously demonstrated that Wwp2 has ubiquitin E3 ligase activity (42
). To investigate whether the CTD of Rpb1 can also serve as a substrate for Wwp2, an in vitro ubiquitination assay was performed with purified proteins including GST-Wwp2, GST-Wwp2CA, GST-CTD, His-ubiquitin, E1, E2 (UBcH6), and GST. When ubiquitination reaction products were analyzed by Western blotting with an anti-ubiquitin antibody, higher-molecular-weight species indicative of the addition of ubiquitin moieties to GST-CTD were observed in the presence of E1, E2, ubiquitin, and wild-type Wwp2 (Fig. , left panel, lane 5). These ubiquitinated products were GST-CTD dependent, since the ubiquitination signal was not seen in the absence of CTD (lane 6). In addition, the ubiquitination signal disappeared when wild-type Wwp2 was replaced with Wwp2CA, demonstrating that Wwp2-mediated CTD ubiquitination was dependent upon an intact HECT domain (lane 7), although mutation of the HECT domain did not affect the interaction between Wwp2 and Rpb1, as demonstrated in Fig. . To further confirm that the ubiquitinated protein was indeed CTD, the same ubiquitination assay was performed, and the reaction products were examined by Western blotting with anti-Rpb1 antibody (C21). Similarly, the higher-molecular-weight signals were detected in the presence of E1, E2, ubiquitin, wild-type Wwp2, and CTD (Fig. , right panel, lane 5) and were absent when GST-CTD was replaced with GST (lane 6). Again, Wwp2CA did not ubiquitinate CTD (lane 7). These data strongly demonstrate that CTD can serve as a substrate for Wwp2-mediated ubiquitination in vitro and that its ubiquitination requires normal activity of all three enzymes involved in the ubiquitination reaction. Of note, the GST-CTD used in the ubiquitination assay was not modified by phosphorylation. Therefore, Wwp2-mediated ubiquitination of CTD is not dependent upon its phosphorylation.
FIG. 3. Wwp2 ubiquitinates Rpb1 both in vivo and in vitro. (A) Wwp2 targets CTD of Rpb1 for ubiquitination in vitro. Various purified proteins as indicated were incubated in ubiquitination buffer. Ubiquitinated CTD of Rpb1 was visualized by Western blotting with (more ...)
The biological relevance of in vitro assays was further investigated by performing in vivo ubiquitination assays. HEK 293 cells were transiently transfected with expression vectors as indicated. Cell lysate was immunoprecipitated with M2 beads and precipitated Rpb1 was analyzed by Western blotting with anti-His antibody. As shown in Fig. , ubiquitination signal was not detected when His-ubiquitin was cotransfected with either Wwp2 or Flag-Rpb1 (lanes 1 and 2, top panel). However, coexpression of His-ubiquitin, Wwp2, and Flag-Rpb1 resulted in Flag-Rpb1 ubiquitination (lane 3). Importantly, the ubiquitin-modified Rpb1 was significantly enhanced when cells were treated with MG132, a 26S proteasome inhibitor, before harvest (lane 4), suggesting that some ubiquitinated Rpb1 is normally degraded through the 26S proteasome. In contrast, we did not detect any ubiquitinated Rpb1 when Wwp2CA was coexpressed (lane 5), although some ubiquitinated Rpb1 was observed when MG132 was used. However, the signal observed with Wwp2CA mutation was much less compared to wild-type Wwp2 (compare lanes 6 and 4) in the presence of MG132. Ubiquitinated Rpb1 observed in lane 6 might result from human WWP2 or other E3 ligases endogenously expressed in HEK 293 cells even in absence of DNA damage. The protein levels for the exogenously expressed Wwp2 were shown in the bottom panel of Fig. . The lower band in Wwp2 Western blot analysis might be degradation product of Wwp2 in HEK 293 cells. These results reveal that Wwp2 can function as an ubiquitin E3 ligase for Rpb1 within cells and provides further evidence for the importance of the cysteine at residue 838 of the HECT domain in the enzymatic reaction.
Having established that Wwp2 can function as an E3 ubiquitin ligase for Rpb1 both in vitro and in vivo, the next question is whether Wwp2 is required for endogenously expressed Rpb1 ubiquitination in physiological conditions. To this end, ubiquitin modification of endogenous Rpb1 in F9 cells expressing Tc-inducible Wwp2 siRNA in the presence of Tc was compared to that in the absence of Tc. The F9 cells were pretreated with 26S proteasome inhibitor to block degradation of ubiquitinated Rpb1, and then Rpb1 in cell lysate was immunoprecipitated with 8WG16 antibody, recognizing hypophosphorylated Rpb1, or H14 antibody, respectively, followed by Western blot analysis with ubiquitin antibody. A high-molecular-weight smear of ubiquitin signal was detected in both 8WG16 and H14 antibody immunoprecipitates when Tc was not present (Fig. ). However, treatment of the F9 cells with Tc dramatically knocked down Wwp2 protein level and, importantly, the level of ubiquitinated Rpb1 simultaneously declined. The Rpb1 protein level was not affected by Tc addition due to the stabilizing effect of MG132 pretreatment. The results provide strong evidence for requirement of Wwp2 to ubiquitinate endogenous Rpb1 protein in normal conditions.
Wwp2 promotes degradation of Rpb1 through 26S proteasome.
The best-studied function of protein ubiquitin modification is its role in protein degradation through the 26S proteasome, although other functions for ubiquitin modification have been discovered. To testify that ubiquitinated Rpb1 catalyzed by Wwp2 can be degraded through 26S proteasome, 26S proteasome degradation assay was performed with in vitro Wwp2-ubiquitinated GST-CTD. As shown in Fig. , GST-CTD fusion protein is modified by ubiquitin in the presence of E1, E2, ubiquitin, and GST-Wwp2 (lane 3). The ubiquitin modification of GST-CTD was dependent on the presence of GST-Wwp2 (no signal in lane 1). Interestingly, the ubiquitinated GST-CTD entirely vanished when 26S proteasome was included in the reaction (lane 4). This result clearly shows that ubiquitinated GST-CTD mediated by Wwp2 was degraded through 26S proteasome.
FIG. 4. Wwp2 promotes degradation of Rpb1 through 26S proteasome. (A) Ubiquitinated GST-CTD catalyzed by Wwp2 can be degraded through 26S proteasome. (B) Knocking down Wwp2 expression elevates the steady-state protein level of Rpb1 in F9 cells. The Tc-inducible (more ...)
The physiological significance of the foregoing observations was tested by studying the role of Wwp2 in regulating Rpb1 protein levels in mouse embryonic pluripotent stem cells. For this purpose, we derived both F9 and CGR8 mouse ES cell lines that stably expressed Tc-inducible siRNAs specifically targeting Wwp2 coding sequences. Two regions in the Wwp2 coding sequence were targeted and are referred to as RNAi Wwp2-1 and RNAi Wwp2-2, respectively. Multiple single clones for each targeted region were selected to ensure the specificity of RNA interference. RNAi EGFP stable F9 cell lines were also generated to be used as Tc treatment control. We induced expression of RNAi Wwp2-1, Wwp2-2, and EGFP by the addition of Tc to cell culture media and compared Rpb1 protein levels in the absence or presence of Tc. Western blot analysis showed that addition of Tc significantly reduced Wwp2 protein level for RNAi Wwp2-2 F9 cell line (Fig. , middle row, lanes 3 to 8). Strikingly, total Rpb1 protein levels in the cells, detected by N20 antibody, were inversely enhanced after addition of Tc (lanes 3 to 5, top row), indicating that the downregulation of Wwp2 elevated Rpb1 protein levels. In contrast, addition of the same dosage of Tc to RNAi EGFP F9 cells affected neither Wwp2 protein levels nor total Rpb1 protein levels (Fig. , lanes 1 and 2). These results indicate that elevated Rpb1 protein levels were a result of reduced Wwp2 levels and not the addition of Tc. Of interest, Rpb1 protein levels were much higher when cells were pretreated with MG132, regardless of the absence or presence of Tc (Fig. , lanes 6 to 8, top row). This phenomenon implies that Rpb1 protein in F9 cells is subject to degradation through the 26S proteasome under normal conditions.
The relationship between Wwp2 and Rpb1 was further assessed by using Rpb1 antibodies specifically recognizing the hyperphosphorylated forms, H14 and H5. Intracellular Wwp2 protein levels gradually decreased after induction of RNAi expression in both RNAi Wwp2-1 and RNAi Wwp2-2 F9 cells, and the reduction in Wwp2 levels was more evident with longer induction (Fig. ). Meanwhile, protein levels for H14 and H5 antibody-recognized hyperphosphorylated Rpb1 were increased. The similar result was obtained with N20 antibody. These results suggest that Wwp2 may promote degradation of Rpb1 in F9 cells and that the regulatory effect of Wwp2 on Rpb1 levels was not dependent upon its phosphorylation state.
We next sought to determine whether Wwp2 has a similar role in the maintenance of steady-state Rpb1 protein level in another cell line. CGR8 ES cells stably expressing Tc-inducible Wwp2 siRNA were used for this purpose. Seven repeats of Wwp2-1 sequence were inserted into the siRNA expression vector to enhance the efficiency of knocking down Wwp2 expression, while only single sequence was used for Wwp2-2 siRNA expression vector. Similarly to F9 cells, Tc treatment induced significant reduction in Wwp2 protein levels in both Wwp2-1(x7) and Wwp2-2 CGR8 ES cell lines. Inversely, H5 antibody- and H14 antibody-recognized hyperphosphorylated Rpb1 and C21 antibody-recognized hypophosphorylated Rpb1 protein levels were gradually upregulated after Tc addition (Fig. ). In contrast, protein level of tubulin remained unchanged with Tc treatment. These data indicate that Wwp2 plays an important role in maintaining Rpb1 protein level at a normal state in embryonic pluripotent stem cells.
Identification of lysine residues in Rpb1 that accept ubiquitin.
It is currently unknown which lysine residues in mammalian Rpb1 are modified by ubiquitination. Therefore, we were keen to identify such modification sites in the GST-CTD fusion protein, which has been demonstrated to be ubiquitinated by Wwp2 in the present study. To this end, we performed mass spectrometric analysis on reaction products from a ubiquitination assay of GST-CTD by Wwp2. Six peptides containing ubiquitin-modified lysine residues were identified (Fig. , the modified lysine residues were italicized and labeled with an asterisk). These were lysine residues at 1859, 1866, 1873, 1887, 1908, and 1922 of murine Rpb1.
FIG. 5. Identification of ubiquitination sites in CTD. (A) Mass spectrometric analysis identified six peptides containing ubiquitinated lysine residues in the CTD. Numbers represent the positions of these peptides in Rpb1. Modified lysine residues were italicized (more ...)
To convincingly prove that these lysine residues are ubiquitin acceptor sites for Wwp2-mediated Rpb1 ubiquitination, we constructed bacterial expression vector which carried mutation of the six lysine residues to arginine residues (GST-CTDmk). After GST-CTD and GST-CTDmk fusion proteins were expressed bacterially, the GST domain was removed by enzymatic digestion to avoid lysine residues in the GST domain being modified by ubiquitin. The CTD and CTDmk proteins were purified and examined for their ubiquitin modification by Wwp2. For clarity, a ubiquitin (K0) with its all seven lysine residues mutated to arginine residues was used in this in vitro ubiquitination assay. As shown in Fig. , CTD was efficiently ubiquitinated by Wwp2 when all components for the ubiquitination reaction were present (lane 2). However, little, if any, ubiquitination was detected when CTD was replaced by CTDmk (lane 3). Next, GST pull-down experiment was conducted between His-Wwp2 and GST-CTD or GST-CTDmk to verify that GST-CTDmk was able to associate with His-Wwp2. As shown in Fig. , mutation of the six lysine residues in CTD did not affect its interaction with Wwp2. This observation demonstrates that the lack of ubiquitination for CTDmk was a result of mutation of the lysine residues as the ubiquitin acceptor sites but not due to a lack of interaction between the two proteins. Next, we examined the requirement of the six lysine residues in CTD for its ubiquitin modification in HEK 293 cells. Both Flag-tagged CTD and CTDmk were expressed in HEK 293 cells together with Wwp2 and His-tagged ubiquitin. MG132 at 20 μM was added to culture media 8 h before harvest. The ubiquitinated proteins in cell lysate were isolated by nitrilotriacetic acid affinity beads and analyzed by Western blotting with anti-Flag antibody. Exogenously expressed Wwp2 and Flag-tagged CTD in whole-cell lysate were also measured by Western blot analysis. As shown in Fig. , His-ubiquitin modified Flag-CTD was easily detected as a high-molecular-weight smear. In contrast, there was not any ubiquitin modification signal detectable for Flag-tagged CTDmk. Finally, we wanted to know whether mutations of the six lysine residues in the CTD of Rpb1 indeed lead to increases in the steady level of CTD. To this end, the same amount of Flag-tagged CTD or CTDmk was expressed in HEK 293 cells together with increasing amounts of Wwp2. At 48 h after transfection, the steady-state levels of wild-type CTD and mutated CTD in the cells were analyzed by Western blotting. As shown in Fig. , the steady-state protein levels of Flag-tagged CTD were gradually decreased with increasing protein level of Wwp2. However, no significant reduction in CTDmk protein level was detected when Wwp2 expression level was elevated. This result clearly shows that CTD expression level is closely associated with intracellular Wwp2 level. Overall, our data demonstrate that the six lysine residues in the CTD of Rpb1 are essential for the ubiquitination and degradation of CTD mediated by Wwp2 both in vitro and in vivo. Thus, we established, for the first time, that six lysine residues in the CTD of Rpb1 could serve as modification sites for Wwp2-mediated ubiquitination and degradation.