Identification of protein kinases responsible for phosphorylation of Epstein–Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function

Kentaro Kato1,2, Akihiko Yokoyama1, Yukinobu Tohya2, Hiroomi Akashi2, Yukihiro Nishiyama3 and Yasushi Kawaguchi1,3,4

1 Department of Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan
2 Department of Veterinary Microbiology, Graduate School of Agricultural and Life Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
3 Department of Virology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
4 PRESTO, Japan Science and Technology Corporation, Tachikawa, Tokyo 190-0012, Japan

Correspondence
Yasushi Kawaguchi
at Nagoya University Graduate School of Medicine
ykawagu{at}med.nagoya-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Epstein–Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization. Earlier studies have shown that the major site of phosphorylation of EBNA-LP by cellular kinase(s) is a serine residue at position 35 (Ser-35) and that the phosphorylation of Ser-35 is critical for regulation of the coactivator function of EBNA-LP (Yokoyama et al., J Virol 75, 5119–5128, 2001). In the present study, we have attempted to identify protein kinase(s) responsible for the phosphorylation of EBNA-LP at Ser-35. A purified chimeric protein consisting of glutathione S-transferase (GST) fused to a domain of EBNA-LP containing Ser-35 was found to be specifically phosphorylated by purified cdc2 in vitro, while GST fused to a mutated domain of EBNA-LP in which Ser-35 was replaced with alanine was not. In addition, overexpression of cdc2 in mammalian cells caused a significant increase in the phosphorylation of EBNA-LP, while this increased phosphorylation was eliminated if Ser-35 of EBNA-LP was replaced with alanine. These results indicate that the cellular protein kinase cdc2 mediates the phosphorylation of EBNA-LP at Ser-35. Recently, we reported that cdc2 and conserved protein kinases encoded by herpesviruses phosphorylate the same amino acid residue of target proteins (Kawaguchi et al., J Virol 77, 2359–2368, 2003). Consistent with this, the EBV-encoded conserved protein kinase BGLF4 specifically mediated the phosphorylation of EBNA-LP at Ser-35. These results indicate that the coactivator function of EBNA-LP can be regulated by the activity of these cellular and viral protein kinases.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus that is frequently associated with infectious mononucleosis and a variety of human malignancies including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, gastric carcinoma and various lymphomas (Kieff & Rickinson, 2001; Rickinson & Kieff, 2001). In vitro, EBV can readily infect and immortalize human B cells (Kieff & Rickinson, 2001; Rickinson & Kieff, 2001). The resultant lymphoblastoid cell lines (LCLs) carry the entire 180 kbp EBV genome encoding more than 80 viral proteins in an episomal and latent state, express only a limited number of viral proteins, including EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-LP, LMP-1, LMP-2A and LMP-2B, and produce no viral progeny (Kieff & Rickinson, 2001; Rickinson & Kieff, 2001). Among these latency-associated EBV proteins, EBNA-1, EBNA-2, EBNA-3A, EBNA-3C, EBNA-LP and LMP-1 are critical for EBV-induced B-cell immortalization, whereas EBNA-3B, LMP-2A and LMP-2B are not (Cohen et al., 1989; Hammerschmidt & Sugden, 1989; Kaye et al., 1993; Mannick et al., 1991; Marchini et al., 1992; Tomkinson et al., 1993). Lytic (or productive) infection can be induced experimentally by the treatment of latently infected cells with chemical reagents such as phorbol ester and sodium butyrate (Kieff & Rickinson, 2001; Luka et al., 1979; zur Hausen et al., 1978), and following induction of virus replication, abundant transcription of a large portion of the genome, the synthesis of many new viral polypeptides, amplification of viral DNA and release of mature virions are observed (Ben-Sasson & Klein, 1981; Hudewentz et al., 1980; Kieff & Rickinson, 2001; Luka et al., 1979; Ragona et al., 1980; Saemundsen et al., 1980; zur Hausen et al., 1979).

EBV nuclear antigen leader protein (EBNA-LP), the subject of this study, is a latency-associated phosphoprotein that is expressed first, together with EBNA-2, following the infection of B cells with EBV in vitro (Alfieri et al., 1991; Petti et al., 1990). It consists of a multi-repeat domain (W1W2) and a unique C-terminal domain (Y1Y2) and has five regions (CR1–CR5) that are evolutionarily conserved among related primate gammaherpesviruses (Fig. 1A, B) (Peng et al., 2000a). As described above, EBNA-LP is considered critical for EBV-induced B-cell immortalization based on the observation that EBNA-LP mutant viruses show severely impaired transforming activity (Allan et al., 1992; Hammerschmidt & Sugden, 1989; Mannick et al., 1991). However, the mechanisms by which EBNA-LP facilitates EBV-induced B cell immortalization remain to be elucidated. Several groups have shown a number of potential interactions of EBNA-LP with cellular proteins including pRb, p53, the 70 kDa family of heat shock proteins (Hsp70), HS1-associated protein X1, bcl-2, {alpha}- and {beta}-tubulins, Hsp27, HA95, protein kinase A and oestrogen-related receptor 1 (Han et al., 2001, 2002; Igarashi et al., 2003; Kawaguchi et al., 2000; Kitay & Rowe, 1996b; Mannick et al., 1995; Matsuda et al., 2003; Szekely et al., 1993). In LCLs, EBNA-LP is localized to discrete nuclear foci (called ND10) that also contain Hsp70, an antigenically distinct form of pRb and CBP/p300 (Bandobashi et al., 2001; Jiang et al., 1991; Szekely et al., 1995, 1996). The plethora of interactions between EBNA-LP and various cellular proteins imply that EBNA-LP is a multifunctional protein that controls various components of the cellular machinery and that the functions of EBNA-LP in the virus life-cycle result from the sum of these interactions. Whatever the significance of these interactions, it is now generally accepted that a major activity of EBNA-LP is to stimulate EBNA-2-mediated transcriptional activation of viral and cellular genes such as LMP-1 and cyclin D2 (Harada & Kieff, 1997; Nitsche et al., 1997; Sinclair et al., 1994). The mechanism by which EBNA-LP expresses its coactivator function has recently been investigated and several lines of evidence described below provide insight into the biochemical role for EBNA-LP in this function. Thus, we previously demonstrated that the conserved region CR2 (Fig. 1B) is a multifunctional domain mediating self-association, nuclear localization and nuclear matrix association of the protein (Tanaka et al., 2002; Yokoyama et al., 2001a). We also mapped the major sites of phosphorylation of EBNA-LP by cellular kinase(s) to serine 35 (Ser-35) in the W2 repeat region (Fig. 1B) (Yokoyama et al., 2001b). The introduction of amino acid substitutions into CR2 or Ser-35 severely impaired the ability of EBNA-LP to induce the expression of LMP-1 in concert with EBNA-2 in B cells (Yokoyama et al., 2001a, b). The requirement for CR2 and Ser-35 for the functions of EBNA-LP was also reported by Peng et al. (2000b) and McCann et al. (2001). Recently, Han et al. (2001, 2002) reported that HA95 and PKA form a complex with EBNA-LP and modulate the coactivator function of EBNA-LP. Taken together, these observations indicate that the modification, cellular localization and protein complex formation of EBNA-LP are critical for the regulation of its coactivator function.



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Fig. 1. (A) Schematic diagram of the sequence of the EBV genome and location of the EBNA-LP and BGLF4 genes. Upper line, linear representation of the EBV genome. The unique sequences are represented as unique 1 to 5 (U1–U5). The terminal and internal repeats flanking the unique sequences are shown as open rectangles with their designations given above. Lower line, expanded section of the domain encoding EBNA-LP (left) and BGLF4 (right). The polarity and the structures of coding regions of EBNA-LP and BGLF4 are shown. (B) The predicted amino acid sequence of the EBNA-LP isoform containing one W repeat. The corresponding exon structures are indicated above the sequence. The conserved regions (CR1–CR5) defined by Peng et al. (2000a) are indicated below the sequence. The major phosphorylation site identified by Yokoyama et al. (2001b) and a sequence that fits the consensus for phosphorylation by cdc2 are shown by an arrow and rectangle, respectively. (C) Schematic diagrams of the expression plasmid containing Flag epitope-tagged wild-type EBNA-LP or EBNA-LP mutant with a serine->alanine substitution at the major phosphorylation sites. (D) Schematic diagram of the predicted amino acid sequence of BGLF4. The shaded areas represent subdomains I–VI, which are conserved in eukaryotic protein kinases (Smith & Smith, 1989). The invariant catalytic lysine is shown by an arrow.

 
As described above, the phosphorylation of Ser-35 in EBNA-LP has been demonstrated to be critical for its coactivator function. EBNA-LP has been reported to be highly phosphorylated during the G2/M phase of the cell cycle and can be phosphorylated by cdc2 and casein kinase II in vitro (Kitay & Rowe, 1996b). However, it is unknown at present which protein kinase(s) are responsible for the phosphorylation of EBNA-LP at Ser-35. An understanding of the action of EBNA-LP in infected cells requires the identification of these protein kinase(s). In the present study, we have attempted to identify the protein kinase(s) targeting the functional phosphorylation site (Ser-35) of EBNA-LP.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
Spodoptera frugiperda Sf9 cells were maintained in Sf-900 II (Invitrogen) supplemented with 10 % foetal calf serum (FCS). The monkey kidney epithelial cell line COS-7 was maintained in Dulbecco's modified Eagle's medium supplemented with 5 % FCS.

Plasmids.
pGEX-W2, pGEX-W2S35A and pGEX-W1W2 were generated by cloning the EcoRI–NotI fragments of pM-W2(H), pM-W2-S35A(H) and pM-EBNA-LPR1d1(H) (Yokoyama et al., 2001b), respectively, into the EcoRI and NotI sites of pGEX4T-1 (Amersham-Pharmacia) in frame with glutathione S-transferase (GST). The construction of the expression plasmid pME-EBNA-LPR3(F) or pME-EBNA-LPR3SA(F) containing Flag epitope-tagged wild-type EBNA-LP cDNA with three copies of the W repeat domain or a mutant version of Flag epitope-tagged EBNA-LP cDNA in which Ser-35 in each W repeat domain is substituted with alanine (Fig. 1C) has been described elsewhere (Yokoyama et al., 2001b). Here we refer to the gene products of pME-EBNA-LPR3(F) and pME-EBNA-LPR3SA(F) as EBNA-LPR3(F) and EBNA-LPR3SA(F), respectively. The expression plasmid pCMVcdc2 for human cdc2 was kindly provided by S. van den Heuvel (van den Heuvel & Harlow, 1993). Construction of pAcGHLT-BGLF4 (see Fig. 3A) and pME-BGLF4(F) has been described previously (Kato et al., 2001). To generate pAcGHLT-BGLF4K102I (Fig. 3A), the lysine at position 102 of BGLF4 was replaced with isoleucine using a QuikChange site-directed mutagenesis kit (Stratagene) with the oligonucleotide 5'-CAGATAATGCCACGGTCATACTCTATGACTCTGTG-3' and its complementary oligonucleotide, according to the manufacturer's directions. To construct pAS-BHRF1, a fragment containing the entire coding sequence of EBV BHRF1 was amplified using pRcCMV-BHRF1 as a template (kindly provided by G. Chinnadurai). The amplified fragment was digested with EcoRI and SalI and cloned into the EcoRI and SalI sites of pAS2 (Clontech) in frame with the DNA-binding domain of GAL4. pMAL-BHRF1 was generated by cloning the EcoRI–SalI fragment of pAS2-BHRF1 into the EcoRI and SalI sites of pMAL-c (New England BioLabs) in frame with maltose-binding protein (MBP). To construct pMAL-EBNA-LPR1, a fragment encoding the entire coding sequence of EBNA-LP with a single W1W2 repeat was amplified by PCR using pACT-EBNA-LPR1 (Tanaka et al., 2002) as a template. The amplified fragment was digested with EcoRI and XbaI and cloned into the EcoRI and XbaI sites of pMAL-c in frame with MBP.



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Fig. 3. Purification of recombinant BGLF4 proteins. (A) Schematic diagrams of the transfer plasmids pAcGHLT-BGLF4 and pAcGHLT-BGLF4K102I used for construction of the recombinant baculoviruses Bac–GST–BGLF4 and Bac–GST–BGLF4K102I, respectively. A silver-stained gel (B) and an immunoblot (C) of purified GST, GST–BGLF4 or GST–BGLF4K102I from Sf9 cells infected with the recombinant baculovirus Bac–GST (lanes 1 and 2), Bac–GST–-BGLF4 (lanes 3 and 4) or Bac–GST–BGLF4K102I (lanes 5 and 6) are shown. Total cell extracts (lanes 1, 3 and 5) were subjected to affinity chromatography on glutathione–Sepharose and eluted (lanes 2, 4 and 6) as described in Methods. The proteins were separated on denaturing gels and subjected to silver staining (B), or transferred onto a nitrocellulose sheet and reacted with the antibody to GST–BMAL1 (C). Molecular masses (kDa) are shown on the left. (D) Purified GST–BGLF4 (lane 1) or GST–BGLF4K102I (lane 2) was incubated in kinase buffer containing [{gamma}-32P]ATP. GST–BGLF4 in kinase buffer containing [{gamma}-32P]ATP was treated with calf intestinal alkaline phosphatase (CIP) (lane 3). The samples were then separated on a denaturing gel and stained with CBB. Molecular masses (kDa) are shown on the left. (E) Autoradiograph of the gel shown in (D).

 
Generation of recombinant baculoviruses.
Recombinant baculoviruses Bac–GST and Bac–GST–BGLF4 were described previously (Kato et al., 2001). To generate Bac–GST–BGLF4L102I, pAcGHLT-BGLF4K102I was co-transfected with linearized baculovirus DNA BaculoGold (Pharmingen) into Sf9 cells using Lipofectin (Invitrogen) as described previously (Kato et al., 2001). The recombinant baculoviruses were propagated in Sf9 cells.

Purification of recombinant BGLF4 proteins from insect cells.
The GST, GST–BGLF4 or GST–BGLF4K102I fusion proteins were purified from Sf9 cells infected with the recombinant baculoviruses Bac–GST, Bac–GST–BGLF4 or Bac–GST–BGLF4K102I, respectively, as described previously (Kato et al., 2001; Kawaguchi et al., 2003).

Production and purification of MBP or GST fusion proteins expressed in E. coli.
GST fusion proteins were expressed in E. coli BL21 transformed with either pGEX-W2, pGEX-W2S35A or pGEX-W1W2 and purified on glutathione–Sepharose beads (Amersham-Pharmacia) as described previously (Kawaguchi et al., 1997a). MBP fusion proteins were expressed in E. coli XL-1 Blue transformed with either pMAL-BHRF1 or pMAL-EBNA-LPR1 and purified on amylose resin (New England BioLabs) according to the procedure followed for the purification of GST fusion proteins except that PBS containing 1 % Tween-20 was used instead of PBS containing 1 % Triton X-100.

In vitro kinase assays.
Purified MBP fusion proteins or GST fusion proteins captured on amylose or glutathione–Sepharose beads were subjected to in vitro kinase assays. These assays were done to determine whether certain MBP or GST fusion proteins could serve as substrates for cdc2 (New England Biolabs) or GST–BGLF4, as described previously (Kawaguchi et al., 2003).

Phosphatase treatment.
After the in vitro kinase assays, the MBP fusion proteins or GST fusion proteins captured on the amylose or glutathione–Sepharose beads were subjected to phosphatase treatment as described elsewhere (Kawaguchi et al., 2003).

Transfection, metabolic labelling and immunoprecipitation.
COS-7 cells were transfected with appropriate combinations of expression plasmids using the DEAE-dextran method as described preciously (Kawaguchi et al., 2000). The transfected cells were labelled with [32P]orthophosphate (Amersham Pharmacia) and then subjected to immunoprecipitation as described elsewhere (Kawaguchi et al., 1998; Yokoyama et al., 2001b).

Immunoblotting.
The electrophoretically separated proteins transferred to nitrocellulose sheets were reacted with appropriate antibodies as described previously (Kawaguchi et al., 1997b, 2000, 2001).


   RESULTS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cellular protein kinase cdc2 targets the functional phosphorylation site Ser-35 of EBNA-LP in vitro
Previous studies provided the following clues about the protein kinases mediating the phosphorylation of EBNA-LP at Ser-35: (i) The sequence flanking Ser-35 (SPTR) (Fig. 1B) completely matches the consensus phosphorylation site for cdc2 [(S/T)PX(KR); Marin et al., 1992]. (ii) EBNA-LP can be phosphorylated by cdc2 in vitro (Kitay & Rowe, 1996a). (iii) In EBV-infected cells, EBNA-LP is highly phosphorylated in the G2/M phase, when the activity of cdc2 is specifically up-regulated (Kitay & Rowe, 1996a; Smits & Medema, 2001). These clues led us to hypothesize that cdc2 phosphorylates Ser-35 of EBNA-LP.

To test this hypothesis, we expressed chimeric proteins consisting of GST fused either to the W2 domain of EBNA-LP (GST–W2) containing Ser-35, to a mutated W2 domain in which Ser-35 was substituted with alanine (GST–W2S35A), or to the W1W2 domain (GST–W1W2) in E. coli transformed with pGEX-W2, pGEX-W2S35A or pGEX-W1W2, respectively (Fig. 2A). GST alone and these GST fusion proteins captured on glutathione–Sepharose beads were used as substrates for in vitro kinase assays in the absence or presence of purified cdc2.



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Fig. 2. The cellular protein kinase cdc2 phosphorylates EBNA-LP at Ser-35. (A) Schematic representation of GST and GST fusion proteins containing either the wild-type W2 domain of EBNA-LP (GST–W2), a mutant of the W2 domain with a substitution of Ser-35 with alanine (GST–W2S35A) or the wild-type W1W2 domain of EBNA-LP (GST–W1W2). The level of phosphorylation of each fusion protein by cdc2 is also shown. (B) Purified GST (lanes 1 and 5), GST–W2 (lanes 2 and 6), GST–W2S35A (lanes 3 and 7) or GST–W1W2 (lanes 4 and 8) was incubated in kinase buffer containing [{gamma}-32P]ATP in the absence (lanes 1–4) or presence (lanes 5–8) of purified cdc2, then separated on a denaturing gel and stained with CBB. Molecular masses (kDa) are shown on the left. (C) Autoradiograph of the gel shown in (B). (D) Purified GST–W2 incubated in kinase buffer containing [{gamma}-32P]ATP and purified cdc2 was treated with alkaline phosphatase (CIP) (lane 1) or mock-treated (lane 2), separated on a denaturing gel and stained with CBB. (E) Autoradiograph of the gel described in (D). (F) Quantification of the relative amount of radioactivity in 32P-labelled EBNA-LP (EBNA-LPR3) or its mutant (EBNA-LPR3LPSA). COS-7 cells were transiently transfected with the indicated expression vectors, labelled with [32P]orthophosphate for 4 h, and then harvested, solubilized, immunoprecipitated with anti-Flag monoclonal antibody (M2) and electrophoretically separated in duplicate on two denaturing gels. Each gel was subjected to immunoblotting with anti-Flag monoclonal antibody (M2) or to autoradiography. The relative amounts of phosphorylated EBNA-LP in autoradiographs were normalized with those of EBNA-LP in immunoblots.

 
As shown in Fig. 2(C), electrophoretically separated GST–W2 or GST–W1W2 was labelled with [{gamma}-32P]ATP in the presence of purified cdc2 (Fig. 2C, lanes 6 or 8), whereas GST or GST–W2S35A was not (Fig. 2C, lanes 5 or 7). Neither GST alone nor any GST fusion protein was labelled in the absence of cdc2 (Fig. 2C, lanes 1–4). The expression of GST and each of the GST fusion proteins and the identity of the radiolabelled band of GST–W2 or GST–W1W2 was verified by Coomassie brilliant blue (CBB) staining (Fig. 2B).

To examine whether the labelling of GST–W2 with [{gamma}-32P]ATP in the presence of cdc2 was due to phosphorylation, the labelled GST–W2 was boiled to inactivate kinases and then incubated with alkaline phosphatase. As shown in Fig. 2(E), the labelling of GST–W2 by incubation with cdc2 was eliminated by phosphatase treatment, indicating that GST–W2 was labelled with [{gamma}-32P]ATP by phosphorylation. The elimination of the labelling was not due to the degradation of GST–W2 by boiling or the phosphatase reaction, as shown by the observation that GST–W2 levels did not decrease after boiling or phosphatase treatment (Fig. 2D). Taken together, this series of experiments indicates that cdc2 protein kinase specifically phosphorylates Ser-35 of EBNA-LP in vitro.

Cdc2 protein kinase mediates the phosphorylation of Ser-35 in EBNA-LP at the cellular level
To test whether cdc2 protein kinase phosphorylates Ser-35 of EBNA-LP at the cellular level, we examined the effect of cdc2 in co-transfection assays with wild-type EBNA-LP or its mutant in which Ser-35 in each W repeat domain was substituted with alanine (Fig. 1C). COS-7 cells transfected with appropriate combinations of expression plasmids using the DEAE-dextran method were labelled with [32P]orthophosphate. Immunoprecipitates obtained by incubation of a mouse monoclonal antibody to Flag epitope (M2; Sigma) with the labelled lysates were solubilized, electrophoretically separated in a denaturing gel and subjected to immunoblotting with anti-Flag antibody (M2) and autoradiography. The bands of immunoblots and autoradiographs were quantified with an image analyser, LAS-1000, using the software Image Gauge (Fuji Film). The relative amounts of phosphorylated EBNA-LP in autoradiographs were normalized against those of EBNA-LP in immunoblots (Fig. 2F).

Since EBNA-LP is phosphorylated by endogenous cellular protein kinase(s) (Yokoyama et al., 2001b), EBNA-LPR3(F) was labelled with 32Pi even when it was expressed alone (Fig. 2F, line 2). The level of EBNA-LP phosphorylation was reduced when Ser-35 was substituted with alanine (Fig. 2F, lines 2 and 4), but EBNA-LPR3SA(F) was consistently labelled with 32Pi (data not shown) as we previously reported (Yokoyama et al., 2001b).

Overexpression of cdc2 caused a significant increase in the phosphorylation of EBNA-LPR3(F) (Fig. 2F, lines 1 and 2), while this increase in phosphorylation was eliminated if Ser-35 of EBNA-LP was replaced with alanine [EBNA-LPR3SA(F)] (Fig. 2F, lines 3 and 4). These results indicate that cdc2 mediates the phosphorylation of Ser-35 in EBNA-LP at the cellular level.

EBV-encoded protein kinase BGLF4 mediates the phosphorylation of EBNA-LP at Ser-35
Recently, we demonstrated that cdc2 and conserved protein kinases encoded by herpesviruses, including herpes simplex virus type 1 (HSV-1) UL13 and EBV BGLF4, phosphorylate the same amino acid residues of target proteins (Kawaguchi et al., 2003). These observations together with the data obtained here and described above led us to hypothesize that an EBV-encoded conserved protein kinase, BGLF4, targets Ser-35 of EBNA-LP for phosphorylation. A series of experiments was designed to test this hypothesis.

In the first series of experiments, we generated and purified a kinase-negative mutant of BGLF4. We previously developed systems to express large amounts of recombinant BGLF4 in insect cells using a recombinant baculovirus and to obtain highly purified BGLF4 with enzymatic activity (Kato et al., 2001). In the present study, we attempted to test whether BGLF4 phosphorylates EBNA-LP in an in vitro assay using purified BGLF4. However, one might argue that the protein kinase activity detected in such experiments is due to contaminating kinase(s) that are physically associated with GST–BGLF4 or fortuitously pulled down by the affinity resins. To eliminate this possibility as far as possible, we constructed a mutant that had no intrinsic protein kinase activity but has probably retained its overall structure. To this end, we generated a recombinant baculovirus (Bac–BGLF4K102I) expressing BGLF4 fused to GST in which lysine-102 (Lys-102) of BGLF4 was replaced with isoleucine by site-directed mutagenesis. We chose Lys-102 in BGLF4 for the site-directed mutagenesis because of the following observations: (i) Lys-102 in BGLF4 corresponds to an invariant lysine in subdomain II of known protein kinases (Fig. 1D) that is required for kinase activity (Hanks et al., 1988); (ii) mutations of the corresponding lysines in BGLF4 homologues of pseudorabiesvirus (PRV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and HSV-1 were shown to result in the loss or reduction of kinase activity (de Wind et al., 1992; He et al., 1997; Kawaguchi et al., 2003; Kenyon et al., 2001); and (iii) it has been reported that overexpression of BGLF4 in mammalian cells mediates the hyperphosphorylation of viral protein EA-D, while that of a mutant of BGLF4 in which Lys-102 was replaced with glutamine did not (Gershburg & Pagano, 2002).

GST, GST–BGLF4 or GST–BGLF4K102I was purified from Sf9 cells infected with either Bac–GST, Bac–GST–BGLF4 or Bac–GST–BGLF4K102I as described in Methods, electrophoretically separated in denaturing gels and either silver stained (Fig. 3B) or immunoblotted with rabbit antiserum containing anti-GST antibody (Kawaguchi et al., 2001) (Fig. 3C). Purified GST, GST–BGLF4 or GST–BGLF4K102I contained one major purified protein with an Mr of 32 000 (GST) or 78 000 (GST–BGLF4 or GST–BGLF4K102I), respectively (Fig. 3B), which reacted with antiserum containing anti-GST antibody (Fig. 3C). Purified GST–BGLF4 and GST–BGLF4K102I were also subjected to in vitro kinase assays to examine their enzymatic activity. When the purified proteins were incubated with [{gamma}-32P]ATP, the wild-type fusion protein was labelled by autophosphorylation (Fig. 3E, lane 1), while the mutant was not (Fig. 3E, lane 2). Furthermore, the labelling of the wild-type was eliminated by treatment with phosphatase (Fig. 3E, lane 3). The expression of each GST fusion protein and the identity of the radiolabelled band were verified by CBB staining as shown in Fig. 3(D). These results indicate that: (i) the desired GST fusion protein was indeed purified; (ii) the kinase-negative mutant with a single amino acid substitution was obtained; and (iii) Lys-102 in BGLF4 is required for the kinase activity.

Next, we performed in vitro kinase assays using the purified recombinant BGLF4 proteins to test whether EBNA-LP is a BGLF4 substrate. A full-length EBNA-LP with a single W1W2 repeat or BHRF1 fused to MBP was purified from E. coli and purified MBP fusion protein (MBP–EBNA-LPR1 or MBP–BHRF1) captured on amylose resin served as substrate for in vitro kinase assays in the presence of the purified wild-type GST–BGLF4 and the kinase-negative mutant GST–BGLF4K102I. As shown in Fig. 4(B), MBP–EBNA-LPR1 was labelled with [{gamma}-32P]ATP by a reaction with purified GST–BGLF4 (Fig. 4B, lane 2), while MBP–BHRF1 was not (Fig. 4B, lane 1). When the kinase-negative mutant GST–BGLF4K102I was used instead of GST–BGLF4 in in vitro kinase assays, neither MBP–BHRF1 nor MBP–EBNA-LP was labelled (Fig. 4B, lanes 3 and 4). The labelling of MBP–EBNA-LPR1 by incubation with GST–BGLF4 was eliminated by phosphatase treatment (Fig. 4D). The expression of each MBP fusion protein and the identity of the radiolabelled band of MBP–EBNA-LP were verified by CBB staining as shown in Fig. 4(A) and (C). This series of experiments indicates that BGLF4 specifically phosphorylates EBNA-LP in vitro.



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Fig. 4. The EBV-encoded protein kinase BGLF4 mediates the phosphorylation of EBNA-LP. (A) Purified MBP–BHRF1 (lanes 1 and 3) or MBP–EBNA-LPR1 (lanes 2 and 4) was incubated in kinase buffer containing [{gamma}-32P]ATP and purified GST–BGLF4 (lanes 1 and 2) or GST–BGLF4K102I (lanes 3 and 4), separated on a denaturing gel and stained with CBB. Molecular masses (kDa) are shown on the left. (B) Autoradiograph of the gel shown in (A). (C) Purified MBP–EBNA-LPR1 incubated in kinase buffer containing [{gamma}-32P]ATP and purified GST–BGLF4 was treated with alkaline phosphatase (CIP) (lane 1) or mock-treated (lane 2), separated on a denaturing gel and stained with CBB. (D) Autoradiograph of the gel shown in (C).

 
We then examined whether BGLF4 targets Ser-35 of EBNA-LP for phosphorylation. The purified chimeric proteins consisting of GST fused to the W2 domain of EBNA-LP (GST–W2) or to a mutated W2 domain in which Ser-35 was substituted with alanine (GST–W2S35A) (Fig. 5A) served as substrates for in vitro kinase assays in the presence of purified GST–BGLF4 or the kinase-negative mutant GST–BGLF4K102I. As shown in Fig. 5(C), GST–W2 was labelled in the presence of GST–BGLF4 (Fig. 5C, lane 1), whereas GST–W2S35A was not (Fig. 5C, lane 2). In contrast, neither of the GST fusion proteins was phosphorylated in the presence of GST–BGLF4K102I (Fig. 5C, lanes 3 and 4). The expression of each GST fusion protein and the identity of the radiolabelled band of GST–W2 were verified by CBB staining (Fig. 5B). These results indicate that Ser-35 of EBNA-LP is the site of phosphorylation by BGLF4 in vitro.



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Fig. 5. EBV BGLF4 mediates the phosphorylation of EBNA-LP at Ser-35. (A) Schematic representation of GST fusion proteins containing the wild-type W2 domain of EBNA-LP (GST–W2) or a mutant of the W2 domain with a substitution of Ser-35 with alanine (GST–W2S35A). The level of phosphorylation of each fusion protein by BGLF4 is also shown. (B) Purified GST–W2 (lanes 1 and 3) or GST–W2S35A (lanes 2 and 4) was incubated in kinase buffer containing [{gamma}-32P]ATP and purified GST–BGLF4 (lanes 1 and 2) or GST–BGLF4K102I (lanes 3 and 4), separated on a denaturing gel and stained with CBB. Molecular masses (kDa) are shown on the left. (C) Autoradiograph of the gel shown in (B). (D, E) Quantification of the relative amount of radioactivity in 32P-radiolabelled EBNA-LP (EBNA-LPR3) or its mutant (EBNA-LPR3LPSA). Experiments were done exactly as described for Fig. 2(F) except that the BGLF4 expression plasmid was used instead of the cdc2 expression plasmid.

 
Finally, we investigated whether BGLF4 phosphorylates Ser-35 of EBNA-LP at the cellular level. To this end, we performed exactly the same cotransfection assays as described in the experiments of Fig. 2(F) except that the expression vector for BGLF4 was used instead of that for cdc2. The results showed that overexpression of BGLF4 caused a significant increase in the phosphorylation of EBNA-LPR3(F) (Fig. 5D), while this increase in phosphorylation was eliminated if Ser-35 of EBNA-LP was replaced with alanine [EBNA-LPR3SA(F)] (Fig. 5E). These results indicate that BGLF4 mediates the phosphorylation of Ser-35 in EBNA-LP at the cellular level. Taken together, this series of results indicates that EBV-encoded protein kinase BGLF4 targets Ser-35 of EBNA-LP for phosphorylation.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An attempt was made to identify protein kinases responsible for the phosphorylation of EBNA-LP at Ser-35, which is critical for its coactivator function (Yokoyama et al., 2001b). In this report, we show that a cellular protein kinase, cdc2, and the EBV-encoded protein kinase BGLF4 mediate the phosphorylation of the functional serine residue (Ser-35) of EBNA-LP, indicating that one or more of the functions of EBNA-LP may be regulated by the activity of these enzymes. The salient features of this study can be summarized as follows.

cdc2 targets Ser-35 of EBNA-LP for phosphorylation
EBNA-LP is a phosphoprotein whose phosphorylation state is dependent on the cell cycle in latently infected cells (Kitay & Rowe, 1996a; Petti et al., 1990). We previously identified Ser-35 as a major phosphorylation site of EBNA-LP by peptide mapping of purified EBNA-LP using mass spectrometry and mutational analyses of the protein (Yokoyama et al., 2001b). Substitution of Ser-35 with an alanine codon resulted in a substantial reduction in the ability of EBNA-2 to transactivate the expression of LMP-1 in EBV-infected cells, while substitution with glutamic acid, which is known to mimic constitutive phosphorylation, restored the wild-type phenotype, indicating that Ser-35 is a functional phosphorylation site (Yokoyama et al., 2001b). Although it has been reported that multiple sites of EBNA-LP can also be phosphorylated by cdc2 or casein kinase II in vitro (Kitay & Rowe, 1996a), Ser-35 is the only functional site that has been shown to be phosphorylated in vivo and to be critical for the function of EBNA-LP (Yokoyama et al., 2001b). In the present study, we have demonstrated that cdc2 phosphorylates the functional site Ser-35 in EBNA-LP, suggesting that cdc2 regulates the coactivator function of EBNA-LP by phosphorylating Ser-35. Our results are consistent with the published observations that phosphorylation of EBNA-LP in infected cells is maximal at G2/M when the activity of cdc2 is specifically up-regulated and that EBNA-LP is phosphorylated at serine residues only in infected cells (Kitay & Rowe, 1996a; Smits & Medema, 2001).

EBV-encoded protein kinase BGLF4 mediates the phosphorylation of EBNA-LP at Ser-35
Herpesviruses contain viral genes that encode protein kinases (Chee et al., 1989; Chung et al., 1989; McGeoch & Davison, 1986; Smith & Smith, 1989). Among them, the amino acid sequences that encode a subset of the viral protein kinases exemplified by HSV-1 UL13 are conserved in all members of the family Herpesviridae (Chee et al., 1989; Smith & Smith, 1989). Here we refer to these viral protein kinases as CHPKs (conserved herpesvirus protein kinases). Recently we reported that the cellular protein kinase cdc2 and CHPKs phosphorylate the same amino acid residues of target proteins, suggesting that CHPKs mimic cdc2 enzymatically in infected cells. These conclusions are supported by two series of observations. First, cdc2 and CHPKs including HSV-1 UL13 and EBV BGLF4 phosphorylate the same site, Ser-133, of cellular elongation factor 1{delta}, which has been reported to be commonly phosphorylated by CHPKs in herpesvirus-infected cells (Kato et al., 2001; Kawaguchi et al., 1998, 1999, 2003). Secondly, HSV-1 UL13 phosphorylates Ser-209 of the casein kinase II {beta} subunit, which was reported to be targeted by cdc2 for phosphorylation (Kawaguchi et al., 2003; Litchfield et al., 1991). These observations prompted us to examine the possibility that EBV BGLF4 phosphorylates EBNA-LP at Ser-35. In the present study, we obtained evidence that this is in fact the case, further supporting our hypothesis that CHPKs mimic cdc2 in infected cells. One may wonder whether BGLF4 in fact interacts with and phosphorylates EBNA-LP in EBV-infected cells since EBNA-LP has been considered a latency-associated protein (Kieff & Rickinson, 2001), while BGLF4 has been reported to be expressed in lytically infected cells (Gershburg & Pagano, 2002). One possible explanation, based on the functional similarities of herpesvirus conserved gene products, is that BGLF4 can be a component of capsid tegument structures like the other CHPKs (Cunningham et al., 1992; Overton et al., 1992; Stevenson et al., 1994; van Zeijl et al., 1997), and is brought into the infected cells by virions and phosphorylates newly synthesized EBNA-LP, which is expressed first following infection of B cells by EBV (Alfieri et al., 1991). Conceivably, it is beneficial to the virus for the virion-associated protein kinase to be brought into infected cells and to phosphorylate Ser-35 of EBNA-LP to express its coactivator function independent of the condition of target host cells, since in vivo EBV infects resting B cells, where the activity of cdc2, which would mediate the functional phosphorylation of EBNA-LP, is down-regulated (Smits & Medema, 2001).

BGLF4K102I is a kinase-negative mutant
There is one puzzling aspect of the studies reported earlier with regard to the invariant catalytic lysine of BGLF4. Chen et al. (2000) previously found that in BGLF4 an amino acid substitution of Lys-102, which is predicted to be the catalytic site of the protein kinase based on analogy with known protein kinases (Hanks et al., 1988), has no effect on the ability of the protein to autophosphorylate. In contrast, Gershburg & Pagano (2002) demonstrated that wild-type BGLF4, which was overexpressed in mammalian cells, mediated the hyperphosphorylation of a viral protein, EA-D, while site-directed mutagenesis of Lys-102 impaired the ability of the protein to mediate the hyperphosphorylation. Furthermore, several laboratories have demonstrated that the corresponding lysines of the other BGLF4 homologues of herpesviruses, including PRV, VZV, HSV-1 and HCMV, cannot be mutated without loss or reduction of their protein kinase activity (de Wind et al., 1992; He et al., 1997; Kawaguchi et al., 2003; Kenyon et al., 2001). In the present study, we obtained evidence that amino acid substitution of Lys-102 of BGLF4 abolished the ability of the protein to autophosphorylate itself or trans-phosphorylate EBNA-LP. These results indicate that Lys-102 is essential for the activity of BGLF4 and suggest that it corresponds to the invariant catalytic lysine conserved in various protein kinases.


   ACKNOWLEDGEMENTS
 
We thank Dr E. Kieff for EBNA-LP cDNA, Dr G. Chinnadurai for pRcCMV-BHFR1, Dr S. van den Heuvel for pCMVcdc2 and Dr K. Maruyama for pME18S. This study was supported in part by Grants-in-Aid for Scientific Research (Y. K. and Y. N.) and Grants-in-Aid for Scientific Research in Priority Areas (Y. K. and Y. N.) from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan and Japan Society for the Promotion of Science (JSPS). K. K. was supported by JSPS Research Fellowships for Young Scientists.


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Received 25 June 2003; accepted 3 September 2003.