Part I. ERK1/2-mediated Phosphorylation of SHDAg at Serine-177 enhances HDV Replication from Antigenomic RNA to Genomic RNA Through RNA Polymerase II Regulation
3.1 The Kinase of SHDAg Serine 177 Phosphorylation.
The contributionsof phosphorylation to SHDAg function have been addressed by mutating Ser-177 or examining the effectsof kinase inhibitor in HDV replication (4, 17, 39, 73, 75). However, the kinase of SHDAg at Ser-177 is never fully understood. In order to identify the kinase for SHDAg Ser-177 phosphorylation, we used the Scansite database (http://scansite.mit.edu/) to look for the clues for the likely enzymes. The searching at low stringency gave a short list of Proline-directed kinases, including CDC2 (CDK1), CDK5, Erk1/2, and p38, with decreasing probabilities (Table 1).
Since direct interaction is a required step for the catalysis of protein phosphorylation, we thus examined whether SHDAg might associate with the enzymes in the above list.
It has been reported that cystein (Cys) mutation at the phosphorylation target residue may stabilize the in vivo interaction between kinase and substrate (48, 69). Hence, Flag-tagged SHDAg mutants, S177C and S2C, along with the wild-type protein were engineered and expressed in HEK293T cells. The amino acid in the serine-2 of SHDAg is highly conserved in HDV strains, therefore, we use S2C as the control in this experiment.
Western blotting showed that these mutations did not affect the SHDAg amounts
in transfected cells and they could be effectively immunopurified using Anti-Flag antibody (Fig. 1).The Erk1/2 kinase was associated with Flag-SHDAgS177C (Fig. 1, lane 8) but that was not detected in the Flag-SHDAgWT and Flag-SHDAgS2C immunoprecipitates (Fig. 1, lanes 6 and 7). In contrast, the CDC2 (CDK1), CDK5, and P38 kinases were not identified in the immunoprecipitates containing either wild type or mutant SHDAg protein. This result suggests that ERK1/2 kinase may associate with SHDAg in vivo and Ser-177 played a role in SHDAg-ERK1/2 interaction.
3.2 MEK1–Mediated Activation of ERK1/2 Induces the Phosphorylation of SHDAg at Ser-177.
The Ser177-directed interaction of SHDAg by ERK1/2 prompted a possibility that ERK1/2 might be a kinase that could phosphorylate SHDAg at Ser-177. To address this, a constitutively active form of MEK1, HA-AcMEK1, was expressed in HEK293T cells, which catalyzed ERK1/2 phosphorylation and thus activated their enzyme activities in a dose-dependent fashion (Fig. 2A) (79). The protein expression of Flag-SHDAg was not perturbed by either the MEK1 or ERK1/2 activities. The lysates from cells expressing Flag-SHDAg protein with or without coexpression of HA-AcMEK1 were immunoprecipitated with anti-Flag antibody-conjugated resin and the proteins were run with SDS-PAGE (Fig. 2B).
After the gel was stained with Commassie Blue, the protein bands of interest were excised and subjected to mass spectrometric analysis (see materials and methods). The Ser-177 residue is located in the peptide, 161GAPGGGFVPNLQGVPESPFSR181, which was extracted from the in gel tryptic digestion. The non-phosphorylated isoform of
161GAPGGGFVPNLQGVPESPFSR181 have m/z values of 1035.97 and 690.99 for its
doubly and triply charged ions, respectively. This non-phosphorylated peptide was consistently eluted at ~51.2 min in different experiments with or without HA-AcMEK1 coexpression (Fig. 2C). Meanwhile, the phosphorylated peptide has m/z values of 1075.97 and 717.65 corresponded to the doubly and triply charged ions, was eluted closely with the non-modified counterpart at 49 min (Fig. 2D). This close elution property is consistent with our previous observations on many phosphorylated peptides (93). The collision-induced dissociation (CID) spectra of the phosphorylated peptides were identified (data not shown), and confirmed that Ser-177 was the phosphorylated residue within the m/z 1075.97 peptides recorded in our previous study (17).
We examined the mass spectrometric data to quantitatively estimate the extent of Ser-177 phosphorylation in vivo using the extracted ion chromatography (XIC) analysis.
Without over-expression of HA-AcMEK1, (Fig. 2D, panel a), XIC analysis showed the intensity of the Ser-177 phosphorylated was as low as 24 cps. However, in the presence of 0.2 μg pHA-AcMEK1 transfection, the intensity of Ser-177 phosphorylation was increased to 120 cps (Fig. 2D, panel b) and was even heightened up to 180 cps when the MEK1 protein expression was further augmented (Fig. 2D, panel c). The intensity of Ser-177 phosphorylation was enhanced for 7.5-folds increase, when compared to that in the cells without HA-AcMEK1 expression. Taken altogether, these data suggest that MEK1-mdiated activation of ERK1/2 is sufficient to enhance SHDAg phosphorylation at Ser-177 in vivo in a dose-dependent manner.
3.3 ERK1/2–Modulated Phosphorylation of SHDAg at Ser 177 is Specifically Recognized by a pS177 Antibody.
By LC/MS/MS analysis, we found that the phosphorylation level of SHDAg at
Ser-177 is increased with HA-AcMEK1 activation in a dose-dependent manner. To further demonstrate that ERK1/2 activation enhances phosphorylation of SHDAg at Ser-177. We probed lysates through Western blot analysis using an antibody specifically against phosphorylated Ser-177, αpS177. This antibody only targeted phosphorylated SHDAg since the prior treatment of the lysate proteins with λ-phosphatase could completely remove the blotting signals (Fig. 3, lane 4). This antibody did not react with S177A mutant (Fig. 3, lanes 5 and 6), which implicates that Ser-177 phosphorylation is required for its reactivity against SHDAg. More importantly, this antibody could monitor the dose-dependent increase in phosphorylation state of SHDAg protein in response to in vivo MEK1 activation (Fig. 3, lanes 2 and 3). These data verify the effectiveness of this αpS177 antibody in quantitative assessment of Ser-177 phosphorylation of SHDAg protein.
3.4 ERK1/2 Phosphorylates SHDAg at Ser-177 in the in vitro Kinase Assay.
To examine whether ERK1/2 kinases could catalyze Ser-177 phosphorylation of SHDAg, we employed a cell-free in vitro kinase assay using highly purified components to examine the enzyme activity on purified SHDAg proteins. The kinases, Flag-ERK1 and Flag-ERK2, expressed in the cells were first activated by treatment with the 20%
serum activation for 30 min, and then Flag-tagged kinases were purified with immunoprecipitation under more stringent conditions. The purity of the enzymes and substrates was verified by silver staining of the polypeptides present in these in vitro reactions (Fig. 4, panel b). Without expression of cellular Flag-tagged proteins, no phosphorylation signals could be detected by αpS177 antibody. In contrast, either Flag-ERK1 or Flag-ERK2 kinase purified with immunoprecipitation could specifically mediate the in vitro phosphate transfer onto wild type Flag-SHDAg protein in a
ATP-dependent fashion (Fig.4, lanes 6 and 8, panel a). These data strongly suggest that ERK1/2 is sufficient to directly phosphorylate SHDAg at Ser-177 residue in vitro.
Taken together, our results indicate that ERK1/2 are kinases capable of catalyzing phosphorylation of SHDAg at Ser-177 both in vivo and in vitro.
3.5 The Activity of HDV Replication from Antigenomic RNA to Genomic RNA is Enhanced by ERK1/2 Kinase Activity Induced by HA-AcMEK1.
We have established that ERK1/2 can catalyze the in vitro and in vivo phosphorylation SHDAg at Ser-177. In previous reports, the Ser-177 residue of SHDAg is crucial for the replication of HDV genomic RNA using the antigenomic RNA as the template (Fig. 5) (73). We wondered whether ERK1/2-mediated Ser-177 phosphorylation might stimulate this RNA replication process.
To test this possibility, we over-expressed constitutively active MEK1 proteins to activate ERK1/2 kinases, and to examine how their activation might affect the HDV production of genomic RNA in a cellular replication system. This system requires the presence of both SHDAg protein and dimeric antigenomic RNA template that is initially transcriptionally synthesized using the pCDm2AG plasmid. The RNA-dependent RNA replication can proceed to both antigenome-to-genome (AG→G) and genome-to-antigenome (G→AG) directions in these cells. It is noteworthy that, although antigenomic RNA can be synthesized in both DNA- and RNA-dependent processes, its amplification is only observed when RNA-dependent replication cycle is established. Thus, antigenomic RNA is usually not detectable in cells only bearing pCDm2AG without the co-expression of SHDAg protein (Fig. 5, lane 3).
In the presence of active ERK1/2 proteins, Ser-177 phosphorylation could be
stimulated dramatically. When more phosphorylated ERK1/2 was present, there existed a significant increase in Ser-177 phosphorylation state in a dose-dependent fashion (Fig.
5 D, lanes 5-7). When MEK1 activity increased, there was a concomitant accumulation of genomic RNA using Northern blot analyses. Paradoxically, the antigenomic RNA was not induced correspondingly when the genomic RNA was more accumulation by ERK1/2 activation in cells (Fig. 5A). These data suggests that activated MEK1-ERK1/2 pathway may increase the efficiency of AG→G replication, while reciprocally decreasing the efficiency of G→AG replication (Fig. 5A, lanes 5-7). Meanwhile, genomic RNA was not detectable when S177A SHDAg mutant was introduced. This is consistent with the notion that this mutant cannot provided the functional activity required for AG→G replication (73).
We also examined the differential effects of MEK1-ERK1/2 pathway on the two directions in the replication cycle using the transcriptionally synthesized genomic RNA as the initiator templates. In the cells expressing pCDm2G plasmid, a similar reciprocal change was seen in the expression of antigenomic and genomic RNAs. The genomic RNA accumulated as MEK activity increased, whereas there was a dose-dependent decrease in antigenomic RNA amount (Fig. 6A, lanes 5-7). As expected, little HDV replication was observed in the system complemented with S177A SHDAg protein (Fig.
6A, lanes 8-10). In accordance with the above results, these observations strongly suggest that MEK1-ERK1/2 pathway, probably through SHDAg Ser-177 phosphorylation, has differential effects on AG→G and G→AG replications.
3.6 The U0126 Reduce the Phosphorylation Level of Ser-177 and Inhibit HDV Replication Activity on Antigenomic RNA to Genomic RNA.
To further confirm the role of ERK1/2-dependent pathway in HDV replication from antigenomic RNA to genomic RNA, we had performed DNA-free HDV antigenomic RNA transfection system. To do this, the pHA-AcMEK1 and pCDSHDAg plasmids were co-transfected with DNA-free dimeric AGm RNA into 293T cells. We blocked the ERK1/2 activities by pharmacological inhibition drug and examined the DNA-free HDV antigenomic RNA replication. As shown in our above result, the HA-AcMEK1 induced high level expression of pERK1/2 and pSer-177 (Fig.7, lane 1). We found that HA-AcMEK1-activated phosphorylation of ERK1/2 was abolished by UO126 (U0126, MEK1 inhibitor), which was accompanied by a decrease in the Ser-177 phosphorylation (Fig. 7, lane 2) as compared to the cells without U0126 treatment (Fig. 7, lane 1). Under these experimental condition, the genomic RNA accumulation from AG to G replication in the cells was reduced (Fig.7, lanes 4). Since the role of the MEK1-ERK1/2 pathway was examined under constitutive MEK1 overexpression conditions, it is not clear whether our findings are physiologically relevant. It would be interesting to know whether U0126 has any effect on Ser177 phosphorylation and HDV replication without MEK1 overexpression. The experiment shown in Figure 8 was performed after prior treatment of cells with UO126 reagents that inhibit the MEK1–ERK1/2 pathway in Huh7 cells. U0126 treatment decreased Ser-177 phosphorylation to the lower level in a dose-dependent manner (Fig. 8A, lanes 3 and 4). The low concentration of 1 μM U0126 did not inhibit HDV replication (Fig.
8B, lane 3), although 10 μM U0126 decreased HDV (Fig. 8B, lane 4). The U0126 studies suggest that the MEK1–ERK1/2 MAPK pathway regulates HDV AG→G replication by modulating the phosphorylation level of SHDAg at Ser-177.
3.7 The Serine 177 is a Critical Amino Acid for Interacting with RNA Polymerase
II.
In previous studies, the Ser177 is phosphorylated and important for HDV replication from antigenomic RNA to genomic RNA (73, 75). The AG to G replication mechanism is α–amanitin sensitive (15, 68). The cellular RNA polymerase II is a α–amanitin sensitive DNA-dependent RNA polymerase. However, many papers showed that RNA polymerase II can help HDV RNA replication in vitro without the DNA template (13, 26, 59, 102, 103). The Ser-177 within the C-terminal region of SHDAg is associated with RNAPII (102). It is possible that SHDAg phosphorylation at Ser177 may play a role in HDV replication on genomic RNA synthesis through regulating the RNAPII.
To study this, we first investigated the role of Ser177 residue in SHDAg-RNAPII complex. We genetically generated a series N-terminal Flag-tagged SHDAg serine mutation constructs, termed Flag-SHDAg S177A (serine 177 change to alanine for mimic the non-phosphorylation form), Flag-SHDAg S177C (serine 177 to cysteine), Flag-SHDAg S177D (serine 177 to aspartic acid for minic the phosphorylation form), Flag-SHDAg S2A (serine 2 change to alanine), and Flag-SHDAg S2C (serine 2 change to cysteine). The amino acid in the Ser-2 of SHDAg is highly conserved in HDV strains, and we used S2C and S2A as the controls in this experiment. The large delta antigen shares the same amino-acid sequences, except for the additional 19-amino acid extension in the C termini. The Flag-LHDAg was also constructed and examined in the experiment. All the pFlag-HDAg plasmids were transiently introduced into 293T cells, and the RNAPII was co-immunoprecipitated with anti-FLAG M2 antibody-bound resin (Sigma) to examine the RNAPII-HDAg associations. The endogenous RNAPII was efficiently recruited by Flag-SHDAg, Flag-SHDAg S2C, and Flag-LHDAg immune-complexes (Fig. 9A, lanes 2, 3 and 5). However, the endogenous RNAPII was
not recruited with Flag-SHDAg S177C as the wild type SHDAg did (Fig 9A, lane 4).
The RNA polymerase I, RPA135, can be recruited with the Flag-SHDAg even the mutagenesis in serine 177 amino acid (Fig 9A, lane 4). This raised an interesting possibility that active turnover of S177 phosphorylation is required for the interaction, since both the mimic for non-phosphorylated and phosphorylated S177 did not associate with RNAPII. The ERK1/2 was also co-immunoprecipitated with Flag-SHDAgS177C (Fig 9A, lane 4). Both of immunoprecipitated RPA135 and ERK1/2 could be the positive control in the experiment.
There are many studies showing that a mutation to aspartic acid can mimic the serine phosphorylation. The FSHDAgS177D mutant, like the FSHDAgS177A, did not recruit endogenous RNAPII (Fig. 9B, lane 10). The aspartic acid may not mimic phosphorylation on SHDAgS177. We also investigate the SHDAg-RNAPII complex without flag-tagged under HDV replication condition (Fig. 9C). We made cell extract after 3 days transfection, and using polyclonal rabbit antibody specific for SHDAg. In the SHDAg S177A mutation co-immunoprecipitation complexes, the associated amount of RNA polymerase II was decreased in Western-blotting (Fig. 9C, lane 6). The similar result was observed with transiently expressed SHDAg in the absence of HDV replication (data not shown). Taken together, it indicates that the Ser-177 residue is critical for the association of HDAgs and Pol II in vivo.
3.8 The Ser-177 Residue is Important for HDV Replication from AG to G.
The amino acid substitution (Ser to Ala, Cys and Asp) of Ser-177 that reduces SHDAg-RNAPII complex stability may abolish HDV replication. We investigated HDV replication in the S177A, S177C, and S177D mutants of SHDAg. The SHDAg mutant
plasmids were co-transfected with pCMV-2AGm plasmid into cells for three days. The Western blotting showed that wild type and mutant SHDAg have the same expression level (Fig.10D). In northern blotting analysis, the S177A, S177C, and S177D mutants do not help the HDV replication (Fig. 10A, lanes 2-4). It shows that Ser-177 residue is critical and neither phosphorylated nor non-phosphorylated analogue alone may facilities HDV replication.
3.9 The Ser-177 Phosphorylation Decreases the Interaction Ability of SHDAg with RNA Polymerase II.
Handa’s group showed that GST-SHDAg purified from E.Coli, supposedly without phosphorylation tag, could interact with mammalian RNAPII nuclear extract in the in vitro binding assay (102). It implies that the initial SHDAg-RNAPII interaction is not
dependent with phosphorylated Ser-177. We should further confirm that the phosphorylation modification on Ser-177 is important for the SHDAg-RNAPII complex.
We propose that ERK1/2 activation or the phosphotase treatment is the relevance of the binding ability of SHDAg to the RNAPII. To study this, the phospho-S177 (ERK1/2 induction) and non-phospho-S177 (phosphotase treating) of SHDAg were immunoprecipitated for analysis RNAPII interaction. The pCMV2-2AG and pHA-AcMEK1 plasmids were transiently expressed in 293T cells. Using immunoprecipitation approach, we investigated the interaction ability in the SHDAg–RNAPII complex in vivo (Fig. 11). The total RNAPII and hyper-phosphorylated RNAPII (CTD-Ser-5 phosphorylation) were probed with ab57300 and 4H8 antibodies. The active HA-AcMEK1 increased the phosphorylation of ERK1/2 and SHDAg (Fig. 11A, lane 3), but decreased the complex formation between FSHDAg and RNAPII (Fig. 11B, lane 3).
We studied the non-phosphorylation Ser-177 immune complexes by λ-phosphatase treatment. The SHDAg immune complexes beads were separated into two parts and then washed in binding buffer in the absence of phosphatase inhibitors and resuspended in phosphatase buffer. Parallel samples were incubated with or without λ-phosphatase (Roche) at 37 °C for 30 min. After the λ-phosphatase treatment, the SHDAg-RNAPII complex is still stable in the assay (Fig. 12, lanes 2 and 4). The results indicated that the Ser-177 phosphorylation may regulate the interaction ability of the SHDAg-RNAPII complex.
3.10 SHDAg Accumulates in the Nucleoplasm in the MEK1-Overexpression Cells.
In previous study, we confirmed that the pERK1/2-mediated Ser-177 phosphorylation enhance HDV replication from AG to G. However, we never know which cell compartment is the site for phosphorylated SHDAg accumulation. To analysis this, the pCMV2-SHDAg plasmid was co-transfected with pHA-AcMEK1 into 293T cells. The immunofluoresce assay using rabbit anti-SHDAg antibody showed that SHDAg distributed in the nucleoplasm more than in the nucleoli (Fig. 13). We have used anti-pSer177 antibody in the same assay, but it was not successful. This clue suggested that shuttling between nucleoplasm and nucleoli may be involved in the regulation of HDV replication mediate by Ser-177 phosphorylation.
Part II. The Proteomic Study of SHDAg in Post-Translational Modifications.
3.11 The Purification of Cytoplasm, Nucleoplasm and Nucleoli from HeLa S3 SHDAg Cell Line.
Since post-translational modifications appear to affect the functional properties of SHDAg, a comprehensive investigation of the modifications on SHDAg isolated from cultured cells is required. The dynamic changes in the post-translational modification state of a protein can affect its activity, stability, cellular localization and interacting partners (6, 20, 23, 24, 53, 54). Both the nucleolus targeting and nuclear speckle localization are required for HDV replication. We thought that the HDV replication in the different cell compartments has the variant PTMs patterns on SHDAg. The phosphorylation, methylation and acetylation on viral antigen may shuttle between cell compartments and recruit the cellular protein for processing the HDV replication. Our goal is to identify the PTM patterns of SHDAg from different nuclear compartments and use mass spectrometry analysis approach to analyze post-translational modifications of SHDAg. To do this, we set up a method for purifying the cytoplasm, nucleoplasm and nucleoli fractions from cells (Fig. 14). The purification method is modified by the proteomics groups (1, 83). All the protein fractions (cytoplasm, nuclei, nucleoplasm and nucleoli) extracted from Hela S3 SHDAg cell lines wereanalyzed by SDS-PAGE and then commasie blue staining (Fig. 15A). The nucleolus B23 (37KD) is concentrated in the nucleoli fraction (Fig. 15A, lane 5, arrow indicated). The histone protein was purified in the nuclear, nucleoplasm and nucleoli fractions, but it was not detectable in the cytoplasm fraction. The Western blotting showed that fibrillarin, a nucleolus protein,
was highly enriched in the nucleoli fraction but scarce in the nucleoplasm fraction (Fig.
15B, lanes 4 and 5). The effective purification of nucleoli could be verified closely with light microscopy (Fig. 15C). In the Figure 16, the total protein concentration and SHDAg purity in different cell compartments was analyzed. The SHDAg is abundant in the nucleolar fraction that is correlated with the nucleolar accumulation patterns in the previous study. These results show that we can effectively purify cytpsolic, nucleoplasmic and nucleolar fractions from the Hela S3 SHDAg cell line. Next, we purified SHDAg from the nucleolar and nucleoplasmic fractions using cation chromatography (Fig 17 and 18).
3.12 Purification of SHDAg with CM Cation Chromatography.
We purified the SHDAgfrom nucleoli extracts of HeLa S3 SHDAg cell by cation chromatography (Fig. 17A). As shown in Fig.17A, sample was loaded into cation column and washed with 20ml buffer A to flow through the unbound proteins. The SHDAg and cellular protein were cofractionated with the gradient elution buffer (buffer A with 0 to 0.5M NaCl and then eluted by buffer A with 0.5M and 1M NaCl). In the western blotting analysis, wefound that SHDAg was eluted in 51 and 52 fractions with
We purified the SHDAgfrom nucleoli extracts of HeLa S3 SHDAg cell by cation chromatography (Fig. 17A). As shown in Fig.17A, sample was loaded into cation column and washed with 20ml buffer A to flow through the unbound proteins. The SHDAg and cellular protein were cofractionated with the gradient elution buffer (buffer A with 0 to 0.5M NaCl and then eluted by buffer A with 0.5M and 1M NaCl). In the western blotting analysis, wefound that SHDAg was eluted in 51 and 52 fractions with