Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27 - PubMed (original) (raw)
Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27
Y Zhi et al. J Virol. 1999 Apr.
Abstract
The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167-182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.
Figures
FIG. 1
Immunoprecipitation of [35S]methionine- or 32Pi-labeled ICP27 from nuclear extracts of HSV-1-infected cells. RSF cells were either mock infected or infected with wild-type HSV-1 strain KOS. Cells were labeled with [35S]methionine (A) or 32Pi (B) in vivo for 5 h, after which the cells were harvested and nuclear extracts were prepared. The extracts were immunoprecipitated with anti-ICP27 monoclonal antibodies H1113 and H1119. The antigen-antibody complexes were separated on an SDS-polyacrylamide gel and detected by autoradiography. The bands corresponding to full-length ICP27 are indicated by arrowheads. The positions of protein molecular weight markers are shown on the left (in kilodaltons).
FIG. 2
Phosphoamino acid analysis of ICP27. RSF cells were infected with KOS and labeled with 32Pi beginning 50 min after infection. After labeling for 3 h (A), 5 h (B), or 13 h (C), the cells were harvested and nuclear extracts were prepared. ICP27 protein was isolated by immunoprecipitation and fractionation on an SDS-polyacrylamide gel. ICP27 was eluted from gel slices and subjected to HCl hydrolysis. The hydrolysates were analyzed by electrophoresis in pH 1.9 buffer in the first dimension and in pH 3.5 buffer in the second dimension. Unlabeled phosphoamino acid standards were stained with 0.25% ninhydrin, and their positions are labeled as follows: S, phosphoserine; T, phosphothreonine; and Y, phosphotyrosine. Labeled phosphoamino acids were detected by autoradiography with intensifying screens.
FIG. 3
Two-dimensional tryptic phosphopeptide maps of ICP27 from infected cells labeled in vivo. RSF cells were infected with KOS and labeled with 32Pi in vivo beginning 50 min after infection. Immunoprecipitated ICP27 was fractionated on an SDS-polyacrylamide gel, eluted from the gel slice, precipitated with TCA, and then digested to completion with TPCK-treated trypsin. The peptides were dissolved in pH 4.72 buffer. Electrophoresis was performed from the cathode (right) toward the anode (left) in pH 4.72 buffer for 25 min at 1,000 V and was followed by chromatography from bottom to top in isobutyric acid buffer. The positions of labeled peptides were visualized by autoradiography with intensifying screens. ICP27 was immunoprecipitated from nuclear extracts of infected cells labeled in vivo for 3 h (A), 5 h (B), or 13 h (C); it was also immunoprecipitated from the cytoplasmic fraction of infected-cell extracts that were labeled for 5 h (D). Arrowheads indicate the sample origins on the TLC plates. Major phosphopeptides are numbered 1 to 4.
FIG. 4
Two-dimensional tryptic phosphopeptide mapping of ICP27 from transfected cells. RSF cells were either infected with KOS or transfected with plasmid pCMV-ICP27 expressing ICP27 and labeled with 32Pi for 5 h. (A and B) Immunoprecipitated ICP27 from either a nuclear extract of infected cells (lanes 1), a cytoplasmic extract of infected cells (lanes 2), or a nuclear extract of transfected cells (lanes 3) was resolved on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The position of ICP27 is indicated by arrowheads. (A) The radioactively labeled proteins on the blot were detected by autoradiography. (B) The blot was subsequently probed with anti-ICP27 monoclonal antibodies (αICP27). The dark band seen in all lanes at 55 kDa is heavy-chain immunoglobulin G which reacts with the secondary antibody used in Western analysis and is present because immunoprecipitated proteins were transferred to the membrane. (C) Immunoprecipitated ICP27 from a nuclear extract of transfected cells (lanes 3, panels A and B) was subjected to two-dimensional tryptic phosphopeptide mapping as described in the legend to Fig. 3. The arrowhead indicates the sample origin on the TLC plate. The four major phosphopeptides are identified by numbers.
FIG. 5
Dephosphorylation of ICP27 by AP and subsequent in vitro phosphorylation with purified PKA catalytic subunit, CKII, or PKC. (A and B) ICP27 from KOS-infected RSF cells was immunoprecipitated and bound to protein A-Sepharose beads. The antigen-antibody complexes were either directly resuspended into SDS sample buffer (lanes 1), incubated with phosphatase buffer alone (lanes 2), or treated with AP (lanes 3). The reaction products were analyzed on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was first exposed to film (A) and then probed with anti-ICP27 monoclonal antibodies (αICP27) (B). The heavy-chain immunoglobulin G band, as described in the legend to Fig. 4, can be seen in all lanes in panel B. (C and D) Dephosphorylated ICP27 was subsequently subjected to in vitro kinase reactions in the absence of added kinase (lanes 2, 4, and 6) or in the presence (+) of exogenous PKA (lanes 1), CKII (lanes 3), or PKC (lanes 5). The reaction products were analyzed on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The radioactively labeled proteins on the blot were visualized by autoradiography with intensifying screens (C); the blot was subsequently probed with anti-ICP27 monoclonal antibodies (D). The positions of ICP27 are indicated by arrowheads.
FIG. 6
Two-dimensional phosphopeptide analysis of ICP27 phosphorylated in vitro with purified PKA catalytic subunit, CKII, or PKC. Immunoprecipitated ICP27 from virus-infected cells was first dephosphorylated by AP in vitro, then phosphorylated with PKA (A), CKII (B), or PKC (C) in the presence of [γ-32P]ATP as shown in Fig. 5C, and finally subjected to two-dimensional tryptic phosphopeptide mapping as described in the legend to Fig. 3. Autoradiography was performed with intensifying screens. Arrowheads indicate the sample origins on TLC plates. Major phosphopeptides are identified by numbers, and unique peptides are indicated by asterisks (see the text).
FIG. 7
Phosphorylation of wild-type ICP27 and frameshift mutant N6R in vivo. RSF cells were transfected with plasmids encoding either wild-type (WT) ICP27 or the frameshift mutant N6R. Twenty-four hours later, the cells were infected with 27-LacZ. Labeling with 32Pi was done for 5 h beginning 50 min after infection. ICP27 proteins were immunoprecipitated with anti-ICP27 monoclonal antibodies (αICP27), separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The blot was first exposed to film (A) and then probed with anti-ICP27 monoclonal antibodies (B). The bands corresponding to full-length ICP27 and to mutant ICP27 are indicated by asterisks. (C and D) Phosphopeptides of both wild-type ICP27 (C) and frameshift mutant N6R, encoding ICP27 from amino acids 1 to 163, were generated by treatment with trypsin and resolved on TLC plates as described in the legend to Fig. 3. Major phosphopeptides are identified by numbers.
FIG. 8
Phosphopeptide analysis of ICP27 mutant proteins labeled in vivo. RSF cells were transfected with a series of phosphorylation site-specific mutants. Viral infection with 27-LacZ to boost protein expression, in vivo 32Pi labeling, and immunoprecipitation of ICP27 were performed as described in the legend to Fig. 7. ICP27 proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. (A and B) The blot was first exposed to film (A) and then probed with anti-ICP27 monoclonal antibodies (αICP27) (B). Lanes: 1, wild-type ICP27; 2, CKII consensus site mutant Δ16–18aa, from which residues 16 to 18 were deleted; 3, CKII consensus site mutant Δ44–46aa, from which residues 44 to and 46 have been deleted; 4, PKA consensus site single-mutant S114A, containing a serine-to-alanine substitution at residue 114; 5, PKA consensus site double-mutant S311,334A, containing serine-to-alanine substitutions at residues 311 and 334; 6, PKA consensus site triple-mutant S114,311,334A, containing serine-to-alanine substitutions at residues 114, 311, and 334. The position of ICP27 is indicated by arrowheads. (C to G) Phosphopeptides of each mutant ICP27 protein were generated by treatment with trypsin and resolved on TLC plates as described in the legend to Fig. 3. Arrowheads indicate the sample origins. Major phosphopeptides are identified by numbers. (C) S114A; (D) S311,334A; (E) S114,311,334A; (F) Δ16–18aa; (G) Δ44–46aa.
FIG. 9
A serine-to-alanine substitution in the PKA consensus site at residue 114 within the ICP27 NLS results in less-efficient nuclear import of the mutant protein. Cells were transfected with plasmids expressing FLAG epitope-tagged wild-type ICP27 (A) or mutant S311,334A (B), S114A (C), or D2ΔS5 (D). In addition, cells were transfected with S114A plasmid DNA and FLAG-tagged wild-type plasmid at a 5:1 ratio (E), with S311,334A plasmid and the wild-type plasmid at a 5:1 ratio (F), or with FLAG-tagged wild-type plasmid DNA and S114A plasmid at a 5:1 ratio (G and H). Twenty-four hours after transfection, the cells were infected with 27-LacZ virus in the presence of cycloheximide (100 μg/ml). The cells were incubated in the presence of cycloheximide for 3 h, at which time the cells were washed, fresh medium without cycloheximide was added, and incubation was continued for an additional 3 h, after which the cells were fixed. The treatment with cycloheximide was performed to synchronize the boost of protein expression that occurred following infection with the 27-LacZ virus and thus allow monitoring of protein import. Cells were stained with anti-FLAG monoclonal antibody (A and E) or with anti-ICP27 monoclonal antibody H1119 (B to D and F to H).
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