The catalytic subunit of protein kinase CK2 phosphorylates in vitro the movement protein of Tomato mosaic virus

Yasuhiko Matsushita1, Mayumi Ohshima1, Kuniaki Yoshioka1, Masamichi Nishiguchi2,{dagger} and Hiroshi Nyunoya1

1 Gene Research Center, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan
2 National Institute of Agrobiological Sciences, 2-1-2 Kan-nondai, Tsukuba, Ibaraki 305-8602, Japan

Correspondence
Hiroshi Nyunoya
nyunoya{at}cc.tuat.ac.jp


   ABSTRACT
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The movement protein (MP) of Tomato mosaic virus (ToMV) was reported previously by us to be phosphorylated in vitro by a cellular protein kinase(s) that exhibited several characteristics of casein kinase 2 (CK2). To characterize further this CK2-like cellular kinase, we have cloned cDNAs encoding the CK2 catalytic subunit from tobacco and compared the properties of the recombinant protein with those of the CK2-like cellular kinase. The recombinant CK2 catalytic subunit formed a complex with ToMV MP and phosphorylated it, similar to the CK2-like cellular kinase. Phosphoamino acid analyses of various mutant MPs altered near the C terminus revealed that the recombinant CK2 catalytic subunit phosphorylated serine-261, while the CK2-like cellular kinase phosphorylated both serine-261 and threonine-256. Both kinases were suggested to phosphorylate an additional serine residue(s) in regions other than the C-terminal peptide. The results are consistent with our previous prediction of involvement of CK2 in phosphorylation of ToMV MP.

The DDBJ accession numbers of the sequences reported in this paper are AB077050–AB077052.

{dagger}Present address: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.


   Introduction
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The movement proteins (MPs) of various plant viruses have been shown to bind to viral genome RNA in vitro and guide it to plasmodesmata to achieve cell-to-cell and long-distance movement in host plants (Lucas & Gilbertson, 1994; Ghoshroy et al., 1997). Among various types of MPs, the ‘30K’ superfamily is the best characterized in Tobacco mosaic virus (TMV) and its close relative Tomato mosaic virus (ToMV), in addition to Cucumber mosaic virus (CMV) and Brome mosaic virus (Melcher, 2000).

Phosphorylation of MPs has been reported in TMV (Citovsky et al., 1993; Haley et al., 1995), ToMV (Watanabe et al., 1992; Kawakami et al., 1999; Matsushita et al., 2000), Potato leafroll luteovirus (PLRV) (Sokolova et al., 1997), Turnip yellow mosaic virus (Seron et al., 1996) and CMV (Matsushita et al., 2002). Waigmann et al. (2000) reported a regulatory role for phosphorylation of the TMV MP in plasmodesmal transport. Karpova et al. (1999) suggested the possibility that phosphorylation of TMV MP modulates the translation-repressing ability of the MP. Protein kinases responsible for the phosphorylation of viral proteins have been characterized to some extent (Lee & Lucas, 2001). PLRV MP was shown to be phosphorylated by a protein kinase C (PKC)-like membrane-associated kinase (Sokolova et al., 1997). Atabekov & Taliansky (1990) suggested the possible involvement of protein kinase A (PKA) in the phosphorylation of TMV MP, while Citovsky et al. (1993) reported the phosphorylation of TMV MP by a cell wall-associated kinase.

Kawakami et al. (1999) reported that serine-37 and serine-238 of ToMV MP were the sites of phosphorylation in vivo and that the two serine residues were within a preferable context for casein kinase 2 (CK2) phosphorylation. Recently, we reported that ToMV MP was phosphorylated in vitro by a CK2-like protein kinase, which formed a stable complex with the MP (Matsushita et al., 2000). However, the replacement of serine-37 and serine-238 with alanine did not affect the phosphorylation by the CK2-like cellular kinase in vitro. Instead, the presence of the C-terminal nine amino acids of the MP was important for in vitro phosphorylation (Matsushita et al., 2000). It remained to be clarified whether the C-terminal residues were the phosphorylation target and/or were responsible for formation of the enzyme–substrate complex. To characterize further the CK2-like cellular kinase for ToMV MP, we have cloned the cDNAs for a CK2 catalytic subunit of tobacco. The catalytic properties of the CK2-like cellular kinase were compared with those of the recombinant CK2 using a series of mutant MPs as substrates.


   Methods
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
cDNA cloning of the tobacco CK2 catalytic subunit.
We cloned a tobacco CK2 by a conventional hybridization method using the Arabidopsis cDNA as a probe since we could not amplify the tobacco CK2 by PCR using degenerate primers. The coding sequence of Arabidopsis thaliana CK2 catalytic subunit (Mizoguchi et al., 1993) was amplified from an A. thaliana MATCHMAKER cDNA Library (Clontech) by PCR using a degenerate primer, CK2F01 (5'-CCNAARGANTAYTGGGAYTAYGA-3'), and a specific primer, CK2R05 (5'-TCATTGACTTCTCATTCTGCT-3'). The amplified 960 bp fragment was cloned into the TA cloning vector pCR2.1 (Invitrogen) and the nucleotide sequence was determined. One of the plasmids, pCR-AtCK2F01R05A, containing the Arabidopsis ATCKA2 cDNA (Mizoguchi et al., 1993), was digested with EcoRI and the resultant 1·0 kb fragment was used as a probe to screen a tobacco cDNA library constructed with the {lambda}GEX5 vector (Matsushita et al., 2001) by plaque hybridization, as previously described (Sassa et al., 2001). Lambda DNA purified from the phage lysate of each clone was used for NotI digestion and subsequent self-ligation to generate a plasmid containing the cDNA insert downstream of the ORF of glutathione S-transferase (GST), as previously described (Matsushita et al., 2001). The nucleotide sequences of the positive clones were determined using appropriate primers.

Genomic Southern blot hybridization.
Genomic DNA was isolated from leaves of Nicotiana tabacum cv. Samsun NN by the phenol/SDS method (Kingston, 1997). Genomic Southern blot hybridization was performed as described previously (Matsushita et al., 2001).

Expression plasmids for GST-fused proteins.
For the production of the GST-fused tobacco CK2 catalytic subunit, the 1·2 kb XhoI–NotI fragment of plasmid pCK2Nt24 derived from the phage clone NtCK2a1 (see Results) was inserted between the SalI and NotI sites of pGEX-6P-1 (Amersham Pharmacia) to create pGEX-NtCK2A. This plasmid contained the coding sequence for a CK2 peptide from the fifth residue (arginine) to the C terminus (glutamine).

The plasmids pGEX-30K, pGEX-30KSS and pGEX-6P2-PKA-30K had been constructed previously to express GST-fused ToMV MPs (Matsushita et al., 2000, 2001). To obtain a C-terminal deletion, a DNA fragment encoding ToMV MP with a deletion of the nine C-terminal amino acids was amplified from pGEX-30KSS by PCR using the vector primer pGEX1 (5'-GCAAGCCACGTTTGGTGGTG-3') and a nested primer, MPdA-R01 (5'-CCGCTCGAGTTACTCGGCTTCATCTTCAAT-3'). The amplified 856 bp fragment was digested with NcoI and XhoI, and the resulting 212 bp fragment was used to replace the 519 bp NcoI–XhoI fragment of pGEX-30KSS to construct pGEX-30KSSdC9.

For production of the GST-fused full-length MP with mutations in the nine C-terminal amino acids, we performed a two-step PCR to obtain the 377 bp StuI–XhoI fragments containing the mutations, which were then substituted for the wild-type fragment of pGEX-30KSS. The first PCR involved two independent PCR events using pGEX-30KSS as template to amplify the upstream and downstream fragments in the coding sequence for the GST-fused MP. These two fragments overlapped each other by 12 or 30 bp, thus allowing heteroduplex formation after denaturation and reannealing. The heteroduplex (1469 bp) encompassing a part of the ORF was made double-stranded and used as the template for the second PCR using the vector primers pGEX1 and pGEX5 (5'- ATTTCCCCGAAAAGTGCCAC-3'). The second PCR products were digested with StuI and XhoI to prepare the 377 bp fragment. To carry out the first PCR, the following internal primers were synthesized: MP256A (5'-ATCCGCGACCGATGCCTCGGCTTCATCTTC-3'), MP261WT (5'-TCGGTCGCGGATTCTGATTCGTATTAAATA-3'), MP257A-R (5'-AGAATCCGCGACTGCCGTCTCGGCTTCATC-3'), MP257A-F (5'-GAAGCCGAGACGGCAGTCGCGGATTCTGAT-3'), MP256WT (5'-ATCCGCGACCGACGTCTCGGCTTCATCTTC-3'), MP261A (5'-TCGGTCGCGGATGCAGATTCGTATTAAATA-3'), MP263A-R (5'-ACATATTTAATATGCATCAGAATCCGCGAC-3'), MP263A-F (5'-TCTGATGCATATTAAATATGTCTTACTCAA-3'). Five different combinations of the two independent primer sets were used for the first PCR: pGEX1/MP256A and MP261WT/pGEX5, pGEX1/MP257A-R and MP257A-F/pGEX5, pGEX1/MP256WT and MP261A/pGEX5, pGEX1/MP263A-R and MP263A-F/pGEX5, and pGEX1/MP256A and MP261A/pGEX5, resulting in the constructs pGEX-30KT256A, pGEX-30KS257A, pGEX-30KS261A, pGEX-30KS263A and pGEX-30KT256AS261A, respectively. However, for construction of pGEX-30KS263A, MP-F01 primer (5'-ATTTAGGTAAGGGGCGTTCA-3') was used instead of pGEX1 primer in the second PCR.

For production of the GST-fused short peptides corresponding to the nine C-terminal amino acids of ToMV MP, two oligonucleotides, MP9aaF1 (5'-AATTGCAGCAACGTCGGTCGCGGATTCTGATTCGTATTAA-3') and MP9aaR1 (5'-TCGATTAATACGAATCAGAATCCGCGACCGACGTTGCTGC-3'), were annealed and inserted between the EcoRI and XhoI sites of pGEX-5X-2 (Amersham Pharmacia) to create pGEX-30KC9, which encoded the fusion protein GST–MPC9 with the nine C-terminal amino acids (TSVADSDSY) of ToMV MP. To obtain the coding sequence for the mutant types of GST–MPC9, two pairs of oligonucleotides were annealed and ligated together to clone between the EcoRI and XhoI sites of pGEX-5X-2. To construct the four types of mutant, 12 oligonucleotides were synthesized: MP9Fa256 (5'-AATTGCAGCAGCATCGGTC-3'), MP9Ra256 (5'-ATCCGCGACCGATGCTGCTGC-3'), MP9FbW (5'P-GCGGATTCTGATTCGTATTAA-3'), MP9RbW (5'-TCGATTAATACGAATCAGA-3'), MP9Fa257 (5'-AATTGCAGCAACGGCAGTC-3'), MP9Ra257 (5'-ATCCGCGACTGCCGTTGCTGC-3'), MP9FaW (5'-AATTGCAGCAACGTCGGTC-3'), MP9RaW (5'P-ATCCGCGACCGACGTTGCTGC-3'), MP9Fb261 (5'-GCGGATGCAGATTCGTATTAA-3'), MP9Rb261 (5'-TCGATTAATACGAATCTGC-3'), MP9Fb263 (5'-GCGGATTCTGATGCATATTAA-3') and MP9Rb263 (5'-TCGATTAATATGCATCAGA-3'). The two pairs MP9Fa256/MP9Ra256 and MP9FbW/MP9RbW were ligated together and used for pGEX-30KC9T256A, encoding the nine amino acids ASVADSDSY. Similarly, MP9Fa257/MP9Ra257 and MP9FbW/MP9RbW, MP9FaW/MP9RaW and MP9Fb261/MP9Rb261, and MP9FaW/MP9RaW and MP9Fb263/MP9Rb263 were used for pGEX-30KC9S257A (encoding TAVADSDSY), pGEX-30KC9S261A (encoding TSVADADSY) and pGEX-30KC9S263A (encoding TSVADSDAY), respectively.

Production and purification of recombinant protein.
The production and purification of recombinant proteins were as described previously (Matsushita et al., 2000). In brief, recombinant proteins were produced in Escherichia coli strain XL10-Gold (Stratagene). The names of the expression plasmids and recombinant proteins are listed in Table 1. The recombinant proteins were purified by binding to glutathione–Sepharose 4B beads (Amersham Pharmacia) and stored on the beads suspended in NETN buffer (50 mM Tris/HCl, pH 8·0, 1 mM EDTA, 150 mM NaCl, 0·5 % NP-40) supplemented with 1 mM dithiothreitol (DTT). Where indicated, purified GST-fused proteins were eluted from the beads with 50 mM Tris/HCl (pH 8·0) buffer containing 10 mM glutathione. In some cases, the fusion proteins immobilized on the beads were treated with PreScission Protease (Amersham Pharmacia) to cleave and separate the protein from the GST portion (Table 1).


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Table 1. List of plasmids and recombinant proteins

 
Protein–protein binding assay.
Protein–protein interactions were examined by a binding assay between a 32P-labelled protein probe and a target protein, which was immobilized on a membrane as described previously (Matsushita et al., 2001). For probe preparation, 32P-labelled PKA–MP was extracted from an SDS-PAGE gel with a solution containing 50 mM NH4HCO3, 0·1 % SDS and 2 % 2-mercaptoethanol. The extracted sample was subjected to ultrafiltration through an Ultrafree-MC membrane (Millipore) to replace the solvent with TBS buffer (20 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·05 % Tween 20, 0·2 % Triton X-100). The recovered protein was renatured by a step-wise ultrafiltration using TBS buffers with decreasing concentrations of guanidine.HCl of 6 M, 1·5 M, 0·38 M and 0 M. The renatured probe protein was dissolved in NETN buffer and used for the binding assay.

Preparation of plant cell extract.
Plant cell extract from the tobacco cell line BY-2 (Nagata et al., 1981) was prepared as previously described (Matsushita et al., 2000). Briefly, plant cells were disrupted by sonication and the extract was prepared in PBS containing 1 mM PMSF and 1 mM DTT supplemented with the additional protease inhibitors pepstatin A (1 µg ml-1), aprotinin (2 µg ml-1), chymostatin (0·1 µg ml-1) and leupeptin (0·5 µg ml-1). The extract was diluted with the same buffer to adjust the protein concentration to 1 mg ml-1 for subsequent assays.

Affinity-enrichment of MP-binding CK2-like cellular kinase(s).
The BY-2 cell extract (30 ml) containing 110 mg of the total proteins was passed through a pretreatment column packed with 2 ml (bed volume) of Sepharose 4B beads (Amersham Pharmacia) to remove non-specific Sepharose-binding proteins. The clarified extract was then passed through an affinity column packed with 250 µl (bed volume) of the glutathione–Sepharose beads conjugated with 250 µg recombinant GST-fused MP. The affinity beads were transferred to a microtube and washed three times with 1 ml NETN buffer containing 3 M NaCl and three times with 1 ml NETN buffer. The beads were resuspended in 350 µl NETN buffer and treated with 6 units PreScission Protease. After incubation on a rotator for 18 h at 4 °C, the cleared supernatant containing the complex of the cleaved MP and CK2-like cellular kinase was used for the kinase assay.

Kinase assay and phosphoamino acid analysis.
The kinase assay and phosphoamino acid analysis were performed as described previously (Matsushita et al., 2000). For the simple kinase assays, recombinant or CK2-like cellular kinases were mixed with test proteins and used for the phosphorylation assays in 30 µl kinase buffer (40 mM HEPES, 10 mM MgCl2, 3 mM MnCl2, pH 7·4) with [{gamma}-32P]ATP. The reaction products were directly subjected to SDS-PAGE. For the protein-complex kinase assays, recombinant or CK2-like cellular kinases were complexed with test proteins on glutathione–Sepharose beads and washed with NETN buffer and kinase buffer before the phosphorylation assays. The reaction products were separated by SDS-PAGE and analysed on a BAS-1500 system (Fuji). For phosphoamino acid analysis, proteins phosphorylated with [{gamma}-32P]ATP were separated by SDS-PAGE and blotted on to PVDF membrane. The protein bands were excised and the phosphoamino acids were analysed as described by Kamps & Sefton (1989).


   Results
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Cloning of tobacco cDNAs for the CK2 catalytic subunit
The Arabidopsis cDNA ATCKA2 encoding a CK2 catalytic subunit (Mizoguchi et al., 1993) was amplified by PCR and used as a probe to screen a tobacco cDNA library constructed with mRNA from the leaves of N. tabacum cv. Samsun NN. Three different cDNA clones, NtCK2a1, NtCK2a2 and NtCK2a3 (DDBJ accession nos AB077050–AB077052, respectively), were obtained from 1x105 plaques. The NtCK2a1 cDNA was 1356 bp in length excluding the poly(A) tail and encoded a protein of 333 amino acids. Compared with the NtCK2a1 cDNA, the NtCK2a2 cDNA was 78 and 5 bp longer at the 5' and 3' regions, respectively, while NtCK2a3 cDNA was 201 and 62 bp shorter at the 5' and 3' regions, respectively. The nucleotide sequences of NtCK2a2 and NtCK2a3 contained one and 20 nucleotides differences, respectively, from the corresponding sequence of NtCK2a1. The nucleotide differences caused one and two amino acid differences in NtCK2a2 and NtCK2a3, respectively.

The deduced protein from the NtCK2a1 cDNA showed a high amino acid identity with the CK2 catalytic subunits from other plants: A. thaliana ATCKA1 (93 %) and ATCKA2 (93 %) (Mizoguchi et al., 1993), Zea mays CK2 {alpha}-1 (93 %), CK2 {alpha}-2 (93 %) and CK2 {alpha}-3 (93 %) (Dobrowolska et al., 1991; Peracchia et al., 1999; Riera et al., 2001), and rice CK2 {alpha} (94 %) (Takahashi et al., 2001). It was also homologous to the CK2 subunits from various organisms such as yeast {alpha}' subunit (62 %) (Padmanabha et al., 1990) and human {alpha} subunit (76 %) (Lozeman et al., 1990).

We performed genomic Southern blot hybridization using the 1·2 kb NtCK2a1 cDNA (which has no internal EcoRI or EcoRV sites) as a probe. As shown in Fig. 1, eight EcoRI (1·3–9 kb) and five EcoRV (3·7–20 kb) fragments were detected, suggesting that the tobacco genome has multiple genes related to CK2 catalytic subunits.



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Fig. 1. Genomic Southern blot hybridization analysis of NtCK2a1. Ten µg N. tabacum cv. Samsun NN genomic DNA was digested with EcoRI (lane 1) or EcoRV (lane 2), fractionated through a 1·0 % agarose gel and transferred on to a nylon membrane. The blot was hybridized with the 32P-labelled 1249 bp EcoRV–NotI fragment of pGEX-NtCK2A containing the cDNA insert. Positions of size markers (kb) are shown on the left.

 
Phosphorylation of ToMV MP by recombinant CK2
We reported previously that ToMV MP was phosphorylated in vitro by a cellular protein kinase that had several characteristics of CK2 (Matsushita et al., 2000). Using the tobacco cDNA NtCK2a1, we produced and purified the recombinant CK2 catalytic subunit (GST–CK2). We then tested whether it phosphorylated ToMV MP (GST–MP) in the simple kinase assay as described in Methods. As shown in Fig. 2, GST–MP was phosphorylated with [{gamma}-32P]ATP when incubated with GST–CK2 (Fig. 2, lane 1) and the labelling intensity was decreased by the addition of the CK2-specific inhibitor, heparin (Fig. 2, lane 2). The phosphorylation did not occur in the absence of GST–CK2 (Fig. 2, lane 3). GST–CK2 was also autophosphorylated (Fig. 2, lanes 1 and 4), a reaction that was also sensitive to heparin (Fig. 2, lanes 2 and 5). As shown below, GST alone was not phosphorylated.



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Fig. 2. Phosphorylation of GST–MP with GST–CK2. Purified GST–CK2 and GST–MP were eluted from glutathione–Sepharose beads and used for the simple kinase assay in the combinations indicated. Phosphorylation reactions were performed in the presence (lanes 2 and 5) or absence (lanes 1, 3 and 4) of 10 µg heparin ml-1. The reaction products were subjected to 10 % SDS-PAGE, visualized by Coomassie blue staining and analysed by autoradiography. Positions of molecular mass markers (kDa) are shown on the left. The lower panel shows the amount of the recombinant proteins after staining with Coomassie blue. Arrows indicate the positions of the recombinant proteins.

 
Binding of recombinant CK2 to ToMV MP
In our previous study, the CK2-like cellular protein kinase formed a stable complex with ToMV MP (Matsushita et al., 2000). To determine whether this occurred with the recombinant tobacco CK2 catalytic subunit, we prepared a recombinant CK2 (rCK2) by removing the GST tag from GST–CK2 and used it for the binding assays with 32P-labelled PKA–MP. As shown in Fig. 3, 32P-labelled PKA–MP bound to rCK2 but not to control GST, suggesting specific binding of MP to rCK2.



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Fig. 3. Binding of MP to rCK2. (a) rCK2 and GST were separated by 12·5 % SDS-PAGE and blotted on to a PVDF membrane. After renaturation of the proteins, the blot was probed with 32P-labelled PKA–MP. (b) The protein profile of the gel used in (a) was revealed by Coomassie blue staining. Positions of molecular mass markers (kDa) are shown on the left. Arrows indicate the positions of the recombinant proteins.

 
Phosphorylation of ToMV MP with a C-terminal deletion by recombinant CK2
We have reported previously that phosphorylation of ToMV MP by a cellular protein kinase was completely abolished by deletion of the nine C-terminal amino acids in the MP when examined by the protein-complex kinase assay (Matsushita et al., 2000). To determine whether this occurred with the phosphorylation by the recombinant tobacco CK2 catalytic subunit, we prepared GST–MP and its deletion mutant GST–MPSSdC9, in which nine amino acids had been removed from the C terminus. We also prepared GST–MPC9, in which only the C-terminal short peptide with the nine amino acids of MP was attached to the C terminus of GST. In the protein-complex kinase assay, rCK2 phosphorylated GST–MPSS (Fig. 4, lane 1) but not GST–MPSSdC9, GST–MPC9 or GST (Fig. 4, lanes 2–4). On the other hand, in the simple kinase assay, rCK2 phosphorylated GST–MPSS (Fig. 4, lane 5) and much less intensely GST–MPC9 (Fig. 4, lane 7). Thus, the protein-complex kinase assay with rCK2 gave the same results as reported previously with the CK2-like cellular kinase (Matsushita et al., 2000). These findings indicate that some of the phosphorylation sites used by rCK2 are located in the C-terminal short peptide of nine residues, but that this short peptide alone is not enough for complex formation with the kinase.



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Fig. 4. Phosphorylation of the GST-fused proteins by rCK2. GST–MPSS, GST–MPSSdC9, GST–MPC9 and GST immobilized on glutathione–Sepharose beads were used for the protein-complex kinase assay (lanes 1–4) and the simple kinase assay (lanes 5–8) with rCK2. The reaction products were analysed as described in Fig. 2. Positions of molecular mass markers (kDa) are shown on the left. Asterisks and the arrowhead indicate the positions of the GST-fused proteins and rCK2, respectively. The upper panel shows a schematic representation of the proteins employed. The lower panel shows the amount of GST-fused proteins after staining with Coomassie blue (CBB).

 
Phosphorylation of ToMV MP with a C-terminal deletion by the CK2-like cellular kinase
To determine whether the C-terminal deletion mutants of MP could form a complex with the CK2-like cellular kinase, we used wild-type GST–MPSS, deletion mutant GST–MPSSdC9 and control GST–6P1 (see Table 1) for affinity-enrichment of the CK2-like cellular kinase from plant-cell extract. The wild-type MP cleaved from GST–MPSS was phosphorylated in a complex with the CK2-like cellular kinase (Fig. 5, lane 1). In contrast, MPdC9 and 6P1 (Fig. 5, lanes 2 and 3) cleaved from GST–MPSSdC9 and GST–6P1, respectively, were not phosphorylated. When an excess amount of MP was added before the assays, the additional MP was phosphorylated by the CK2-like cellular kinase enriched by GST–MPSS as well as GST–MPSSdC9 (Fig. 5, lanes 4 and 5). The results suggest that a CK2-like cellular kinase phosphorylation target is located in the nine amino acids of the MP but that this short peptide is not required for complex formation between ToMV MP and the CK2-like cellular kinase.



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Fig. 5. Binding of the CK2-like cellular kinase with the GST-fused proteins. GST–MPSS, GST–MPSSdC9 and GST–6P1 were used for affinity-enrichment of the CK2-like cellular kinase from BY-2 cells. The complex of the cleaved MP and CK2-like cellular kinase was used for the simple kinase assay in the absence (None; lanes 1–3) or presence (+ MP; lanes 4–6) of additional MP. Positions of molecular mass markers (kDa) are shown on the left. Arrows indicate the positions of the recombinant proteins. Asterisks indicate the positions of MP and MPdC9. The upper panel shows a schematic representation of the proteins employed. The lower panel shows the amount of the recombinant proteins after staining with Coomassie blue (CBB).

 
Phosphorylation of ToMV MP with amino acid substitutions in the C-terminal region
In our previous study, ToMV MP was shown to be phosphorylated at both serine and threonine residues by an MP-binding CK2-like cellular kinase in vitro (Matsushita et al., 2000). We introduced single or double alanine substitutions at threonine-256, serine-257, serine-261 or serine-263 in the C-terminal region of ToMV MP. These mutant MPs were used for phosphorylation by recombinant CK2 as well as the CK2-like cellular kinase. We also prepared GST–MPSSdC9 in which the nine amino acids were removed from the C terminus of ToMV MP. In the simple kinase assay with rCK2, the phosphorylation levels of GST–MPSS, GST–MPT256A, GST–MPS257A (Fig. 6a, lanes 1–3) and GST–MPS263A (Fig. 6a, lane 5) were comparable with each other, while those of GST–MPS261A and GST–MPT256AS261A (Fig. 6a, lanes 4 and 6) decreased significantly. The phosphorylation level of GST–MPSSdC9 was still lower (Fig. 6a, lane 7). In the protein-complex kinase assay with the CK2-like cellular kinase, there were no gross effects of the amino acid substitution at serine-261 (Fig. 6b, lanes 4 and 6), while there was a significant decrease in the phosphorylation level of GST–MPSSdC9 (Fig. 6b, lane 7).



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Fig. 6. Phosphorylation of the mutant MPs with amino acid substitutions. The recombinant proteins GST–MPSS, GST–MPT256A, GST–MPS257A, GST–MPS261A, GST–MPS263A, GST–MPT256AS261A, GST–MPSSdC9 and GST–6P1 were immobilized on glutathione–Sepharose beads and used for the simple kinase assay with rCK2 (a) or the protein-complex kinase assay with BY-2 cell extract (b). The reaction products were analysed as described in Fig. 2. Positions of molecular mass markers (kDa) are shown on the left. Arrows indicate the positions of the recombinant proteins. An arrow head indicates the position of rCK2. The lower panels show the amount of the recombinant proteins stained with Coomassie blue (CBB). (c) Phosphoamino acid analysis of the recombinant proteins phosphorylated in (b). The radioactivities of the total phosphoamino acids were adjusted to equivalent levels for electrophoresis. Positions of phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) are shown on the left.

 
Phosphoamino acid analysis revealed that rCK2 phosphorylated serine residues but not threonine or tyrosine residues of GST–MPSS, GST–MPS261A and GST–MPSSdC9 (data not shown). On the other hand, the CK2-like cellular kinase phosphorylated both serine and threonine residues of GST–MPSS and GST–MPS261A (Fig. 6c). As shown in Fig. 6(c), phosphothreonine was not detected when threonine-256 was replaced by alanine (T256A and T256AS261A) or removed together with the other C-terminal residues (dC9). It should be noted, however, that the relative amount of phosphoserine to phosphothreonine decreased in GST–MPS261A (S261A) when compared with those of GST–MPSS (Fig. 6c), GST–MPS257A (data not shown) and GST–MPS263A (data not shown). This indicated that phosphorylation at serine-261 by the CK2-like cellular kinase contributed significantly to the gross phosphorylation level of serine residues in the MP.

In conclusion, both the CK2-like cellular kinase and rCK2 phosphorylated serine-261 and some additional serine residue(s) other than in the C-terminal region of ToMV MP. Since the other serine residue(s) was more intensely phosphorylated by the CK2-like cellular kinase than rCK2, the effect of the S261A substitution was apparently diminished in the protein-complex kinase assay (Fig. 6b). It also appeared that the CK2-like cellular kinase phosphorylated threonine-256 as the only target threonine residue. The recombinant CK2 could not phosphorylate any threonine residues in ToMV MP.

Phosphorylation of the C-terminal peptide of MP by recombinant CK2
To analyse the specific phosphorylation residues, we used the recombinant protein GST–MPC9, in which the short peptide composed of the nine C-terminal amino acids of ToMV MP was fused to GST, and derivatives with alanine substitutions for the previously mentioned threonine or serines (Table 1). As shown in Fig. 7(a), rCK2 phosphorylated GST–MPC9, GST–MPT256A, GST–MPS257A and GST–MPS263A, but not GST–MPS261A or control GST. Phosphoamino acid analysis confirmed that phosphorylation occurred only at the serine residue (Fig. 7b). Although the phosphorylation efficiency of the short peptide was much lower than that of full-length MP (Fig. 4, lanes 5 and 7), serine-261 was confirmed to be one of the targets of CK2. We could not detect any significant phosphorylation when the above GST-tagged peptides were assayed with the affinity-enriched CK2-like cellular kinase (data not shown). The catalytic properties of the possible multimeric CK2-like cellular kinase may depend more strictly on the substrate integrity compared with the simple catalytic subunit of CK2.



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Fig. 7. Phosphorylation of GST-fused C-terminal short peptides by rCK2. (a) Eluted GST–MPC9, GST–MPC9T256A, GST–MPC9S257A, GST–MPC9S261A, GST–MPC9S263A and GST were used for the simple kinase assay with rCK2 as described in Fig. 2. Positions of molecular mass markers (kDa) are shown on the left. The upper panel shows the autoradiogram and the lower panel shows the amount of the recombinant proteins stained with Coomassie blue (CBB). (b) Phosphoamino acid analysis of the corresponding recombinant proteins shown in (a). Positions of phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) are shown on the left.

 

   Discussion
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Protein kinase CK2 is an essential, ubiquitous and highly pleiotropic protein kinase. Its substrate proteins have been implicated in signal transduction, transcriptional control, cell proliferation and various steps of development. Pathogens such as animal viruses and parasites are also reported to utilize the host CK2 to phosphorylate their component proteins (Guerra & Issinger, 1999). The CK2 holoenzyme is composed of two catalytic ({alpha} and/or {alpha}') and two regulatory {beta} subunits forming stable heterotetramers (Guerra & Issinger, 1999). A monomeric enzyme has also been reported in some plants (Dobrowolska et al., 1992). To date, cDNA clones for plant CK2 catalytic subunits have been reported in A. thaliana (Mizoguchi et al., 1993), maize (Dobrowolska et al., 1991; Peracchia et al., 1999, Riera et al., 2001), rice (Takahashi et al., 2001) and tobacco (Espunya et al., 1999).

We have previously shown that the nine C-terminal amino acids of ToMV MP are necessary for in vitro phosphorylation by a protein-complex kinase assay with a CK2-like cellular kinase (Matsushita et al., 2000). However, it remained to be determined whether the nine amino acids were the target sequence or were only required for the complex formation. In the present study, we have shown that both the recombinant CK2 catalytic subunit and the CK2-like cellular kinase can make a stable complex with ToMV MP and that they phosphorylate the same serine residue in the C-terminal region of the MP. Although the C-terminal short peptide of nine amino acids directly attached to GST was not sufficient for the complex formation with rCK2 (Fig. 4, lane 3), the peptide could serve as a substrate for rCK2 (Fig. 4, lane 7). Furthermore, alanine substitution for serine-261 abolished phosphorylation of this peptide (Fig. 7). Since both the wild-type and a deletion mutant lacking the nine amino acids of the MP were shown to bind the CK2-like cellular kinase in affinity-enrichment (Fig. 5), the nine C-terminal amino acids are dispensable for complex formation.

Our in vitro assay failed to demonstrate involvement of the recombinant CK2 catalytic subunit in phosphorylation of threonine-256 (Fig. 7b), which was targeted by the MP-associating CK2-like cellular kinase (Fig. 6c). Another difference between the catalytic subunit and the CK2-like cellular kinase was their relative contributions to phosphorylation of an internal serine residue(s) (Fig. 6a, b, lanes 4 and 6). These discrepancies could be attributed to the existence of the {beta} subunit of CK2, which should be present in the multimeric CK2-like cellular kinase and regulate its enzymatic properties. In this context, it is interesting that phosphorylation of the Rev transactivator protein of human immunodeficiency virus type 1 by CK2 is dependent on the {beta} subunit (Marin et al., 2000). Alternatively, different types of CK2 catalytic subunits could be involved in the phosphorylation of ToMV MP. The occurrence of closely related genes in the tobacco genome was shown in the Southern blotting analysis (Fig. 1). Further experimentation will be required to identify definitively our extract protein as a CK2 kinase.

Although the threonine-256 and serine-261 are not surrounded by the consensus phosphorylation motif S/T-XX-D/E (Pearson & Kemp, 1991), the 14 C-terminal amino acid residues (EDEAETSVADSDSY) including the phosphorylation sites are quite rich in acidic amino acids. According to Meggio et al. (1994), an acidic amino acid residue at position +3 is not an absolute requirement for phosphorylation by CK2, and they reported that additional acidic residues at positions spanning from -2 to +7 act as positive specificity determinants for CK2.

In the closely related TMV, a cell wall-associated protein kinase (Citovsky et al., 1993) and other kinases (Haley et al., 1995) are reported to phosphorylate the serine residues of MP. Since we used a buffer without any detergents to prepare the cell extract, it should contain only soluble material and may not contain any cell wall- or membrane-associated protein kinases. Moreover, we focused on the CK2-like cellular kinase that formed a stable complex with ToMV MP. Therefore, we do not exclude the possibility of the participation of other protein kinases in the phosphorylation of ToMV MP.

Our in vitro results showed that ToMV MP is phosphorylated mostly in the C-terminal region. Watanabe et al. (1992) reported that the 31 C-terminal amino acids of ToMV MP are required for phosphorylation of the MP but the region is dispensable for viral cell-to-cell movement. In TMV MP, the C-terminal amino acids were also reported to be dispensable for viral cell-to-cell movement (Gafny et al., 1992; Boyko et al., 2000). Despite the dispensability of the C-terminal region, phosphorylation at this region was suggested to have a regulatory role in plasmodesmal transport (Waigmann et al., 2000). As Padgett et al. (1996) suggested, the cellular distribution and conformation of the MP may change from early to late stages of virus infection. The phosphorylation status of the MP could depend on these changes, which could affect the accessibility of the MP toward cellular protein kinases and other factors. Such a dynamic aspect of the behaviour of the multifunctional MP protein might explain discrepancies between our in vitro data and the in vivo results reported by Kawakami et al. (1999).


   ACKNOWLEDGEMENTS
 
This work was supported in part by the Grant-in-Aid for Young Scientists (B) to Y. M. (nos. 11760033 and 13760035) and one for Scientific Research on Priority Areas to H. N. and Y. M. (nos. 12052209 and 13039007) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. It was also supported by the Grant-in-Aid ‘Integrated Research Program for the Development of Innovative Plants and Animals Using Transformation and Cloning’ from the Ministry of Agriculture, Forestry and Fisheries of Japan to H. N.


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Received 19 September 2002; accepted 15 October 2002.