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
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ABSTRACT |
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The DDBJ accession numbers of the sequences reported in this paper are AB077050AB077052.
Present address: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.
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Introduction |
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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 enzymesubstrate 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.
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Methods |
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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 XhoINotI 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 NcoIXhoI 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 StuIXhoI 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 GSTMPC9 with the nine C-terminal amino acids (TSVADSDSY) of ToMV MP. To obtain the coding sequence for the mutant types of GSTMPC9, 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 glutathioneSepharose 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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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 glutathioneSepharose 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 [
-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 glutathioneSepharose 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 [
-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)
.
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Results |
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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
-1 (93 %), CK2
-2 (93 %) and CK2
-3 (93 %) (Dobrowolska et al., 1991
; Peracchia et al., 1999
; Riera et al., 2001
), and rice CK2
(94 %) (Takahashi et al., 2001
). It was also homologous to the CK2 subunits from various organisms such as yeast
' subunit (62 %) (Padmanabha et al., 1990
) and human
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 (1·39 kb) and five EcoRV (3·720 kb) fragments were detected, suggesting that the tobacco genome has multiple genes related to CK2 catalytic subunits.
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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 GSTMPC9, 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 GSTMPC9, GSTMPT256A, GSTMPS257A and GSTMPS263A, but not GSTMPS261A 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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Discussion |
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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
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
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)
.
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ACKNOWLEDGEMENTS |
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Received 19 September 2002;
accepted 15 October 2002.