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Journal of General Virology (2000), 81, 2095–2102. Printed in Great Britain ................................................................................................................................................................................................................................................................................... In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase Yasuhiko Matsushita,1 Kohtaro Hanazawa,1 Kuniaki Yoshioka,1 Taichi Oguchi,1 Shigeki Kawakami,2 Yuichiro Watanabe,2 Masamichi Nishiguchi3 and Hiroshi Nyunoya1 1 Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan 3 National Institute of Agrobiological Resources, 2-1-2 Kan-nondai, Tsukuba, Ibaraki 305-8602, Japan 2 The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [γ-32P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [γ-32P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II. Introduction The movement proteins (MPs) encoded by plant viruses have been shown to be essential for cell-to-cell movement through intercellular connections called plasmodesmata (Deom et al., 1987, 1992 ; Meshi et al., 1987, 1992 ; Lucas & Gilbertson, 1994 ; Carrington et al., 1996). MPs are also known to have nonspecific single-stranded nucleic acid-binding activity, suggesting the ability of MPs to bind to and aid in transport of the viral RNA from cell to cell (Citovsky et al., 1990 ; Li & Palukaitis, 1996 ; Fujita et al., 1998). In the case of tobamoviruses such as tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), MPs are known to be synthesized in the early stages of infection (Watanabe et al., 1984) and to be localized in plasmodesmata (Tomenius et al., 1987 ; Atkins et al., 1991 a). MPs are also reported to be involved in host Author for correspondence : Hiroshi Nyunoya. Fax j81 42 367 5563. e-mail nyunoya!cc.tuat.ac.jp 0001-6982 # 2000 SGM specificity, suggesting interactions between MPs and host-cell factors (Taliansky et al., 1982 ; Atabekov & Dorokhov, 1984 ; Meshi et al., 1989 ; Mise et al., 1993 ; Weber et al., 1993 ; Fenczik et al., 1995 ; Weber & Pfitzner, 1998 ; Reichel et al., 1999). Phosphorylation of TMV MP has been examined by several groups. Atkins et al. (1991 b) showed that plantexpressed TMV MP comigrates during SDS–PAGE with the phosphorylated form of a recombinant MP prepared from insect cells infected with baculovirus. Direct evidence for in vivo phosphorylation was obtained by using TMV RNAinoculated protoplasts (Watanabe et al., 1992 ; Haley et al., 1995) or MP-expressing transgenic plants (Citovsky et al., 1993). These groups have identified possible phosphorylation sites in several regions including the serine-rich C-terminal peptide. For example, Citovsky et al. (1993) have demonstrated phosphorylation of serine-258, threonine-261 and serine-265 of TMV MP by a cell wall-associated protein kinase. Kawakami et al. (1999) identified serine-37 and serine-238 as the sites of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 CAJF Y. Matsushita and others phosphorylation in vivo and suggested that the presence and state of phosphorylation of serine-37 in MPs is important for cell-to-cell movement of the virus genome. The protein kinases that phosphorylate MPs have not yet been firmly identified although there are reports on the possible involvement of cyclic AMP-dependent kinase (Atabekov & Taliansky, 1990) and cell wall-associated protein kinase (Citovsky et al., 1993). Since protein kinases are not encoded by plant viruses (Goelet et al., 1982 ; Ohno et al., 1984), candidate kinases may be considered as host factors interacting with MP. As a first step to characterize such hostplant kinases, we prepared a recombinant MP and established a protein-complex kinase assay system. Methods Plasmid construction. The 1n0 kb MaeI fragment of plasmid pLQV5 (Meshi et al., 1992), containing the coding sequence for the MP of ToMV (formerly designated TMV tomato strain L), was treated with Klenow fragment to fill in the 3h termini and inserted into the SmaI site of pBluescriptII SK(j) (Stratagene) to create pBS-30K, with the coding sequence in the EcoRI to NotI sense of the vector. The 1n0 kb EcoRI–NotI fragment was subsequently inserted into the EcoRI–NotI sites of pGEX5X-2 (Amersham Pharmacia) to produce pGEX-30K, which encodes glutathione S-transferase (GST)-fused ToMV MP. The 1n0 kb MaeI fragments of plasmids pTLW3 and pTLQ37A238A (Kawakami et al., 1999), containing the coding sequence for wild-type and mutant ToMV MPs, respectively, were treated with Klenow fragment to fill in the 3h termini and inserted into the SmaI site of pGEX6P-3 (Amersham Pharmacia) to create pGEX-30KSS and pGEX30KS37AS238A, respectively. Both vectors contain the coding sequences in the EcoRI to NotI sense of the vector. pGEX-30KSS encodes GST-fused wild-type MP while pGEX-30KS37AS238A encodes GST-fused mutant MP with alanine residues substituted for serine-37 and serine-238. For construction of the plasmids encoding GST-fused ToMV MPs with C-terminal truncations, plasmid pGEX-30K was digested with AatII alone or StuI plus XbaI to remove the 0n54 kb AatII or 0n31 kb StuI–XbaI fragments, respectively. The remaining larger DNA fragments were treated with Klenow fragment before self-ligation to create plasmids pGEX-30KdA and pGEX-30KdSX. Production of recombinant protein. Recombinant proteins were produced in E. coli strain XL-1 Blue (Stratagene) transformed with the various plasmids constructed for expression of GST fusion proteins. The names of the plasmids and corresponding recombinant proteins were as follows : pGEX-5X-2 for GST ; pGEX-30K for GST–MP ; pGEX-30KSS for GST–MPSS ; pGEX-30KS37AS238A for GST–MPAA ; pGEX-30KdA for GST–MPdA ; pGEX-30KdSX for GST–MPdSX. The recombinant protein GST–MPdA had the C-terminal 9 amino acids replaced by 27 nonviral residues (QVALFGEMCAEPLFVYFSKYIQICIRS) derived from the vector. Another recombinant protein, GST–MPdSX, had the Cterminal 31 amino acids replaced by 7 residues (LERPHRD) derived from the vector. Protein expression was induced by addition of 0n2 mM IPTG. The recombinant proteins were purified using glutathione–Sepharose 4B beads (Amersham Pharmacia) as described by Kaelin et al. (1991) and stored in a modified NETN buffer (50 mM Tris–HCl, pH 8n0, 1 mM EDTA, 150 mM NaCl, 0n5 % Nonidet P-40) supplemented with 1 mM dithiothreitol (DTT). Preparation of plant-cell extracts. Seeds of Nicotiana tabacum L. cv. Samsun NN were germinated and grown under a light (16 h)\dark CAJG (8 h) cycle at 24 mC. Suspension cultures of a BY-2 tobacco cell line were maintained as described by Nagata et al. (1981) and cells in the lateexponential phase were frozen at k80 mC after washing with PBS (137 mM NaCl, 2n68 mM KCl, 10n1 mM Na HPO , 1n76 mM KH PO , # % # % pH 7n4). To prepare the cell extracts, leaves (0n1 g fresh wt\ml) of the tobacco plants and frozen BY-2 cells (0n4 g fresh wt\ml) were suspended in PBS supplemented with 1 mM DTT and 1 mM PMSF, homogenized using a Polytron (PT3000 ; Kinematica) and then disrupted by sonication. After centrifugation for 20 min at 16 000 g, the supernatant was diluted with PBS to adjust the protein concentration to 1 mg\ml for use in the kinase assay. Kinase assay. For the simple kinase assay, glutathione–Sepharose 4B beads conjugated to 1 µg of recombinant protein were suspended in 100 µl of kinase buffer (40 mM HEPES, pH 7n4, 10 mM MgCl , 3 mM # MnCl ) including 45 µl cell extract as prepared above plus the protease # inhibitors pepstatin A (1 µg\ml), aprotinin (2 µg\ml), chymostatin (0n1 µg\ml), leupeptin (0n5 µg\ml) and trans-epoxysuccinyl--leucylamido-[4-guanidino]butane (7n2 µg\ml). The phosphorylation assay was started by addition of 370 kBq [γ-$#P]ATP (168 TBq\mmol), incubated for 30 min at 25 mC on a rotator, and terminated by washing the beads twice with 0n9 ml NETN buffer. For the protein-complex kinase assay, glutathione–Sepharose beads conjugated to 1 µg recombinant protein were incubated in 1 ml PBS containing a 45 µl aliquot of plant-cell extract and protease inhibitors for 1 h at 4 mC on a rotator. The beads thus treated were washed twice with 1 ml of NETN buffer, twice with 1 ml of kinase buffer and resuspended in 100 µl of kinase buffer. The phosphorylation assay was performed with [γ-$#P]ATP under the same conditions as the simple kinase assay, except that no additional cell extract and protease inhibitors were added. For pull-down experiments, diluted plant extracts were preincubated with appropriate beads before protein-complex formation with GST–MP. Protein phosphorylation was analysed by SDS–PAGE followed by image analysis with a BAS-1500 system (Fuji Photo Film). Phosphoamino acid analysis. Proteins phosphorylated with [γ-$#P]ATP were separated by SDS–PAGE and blotted onto PVDF membrane. The protein band was excised and hydrolysed for 2 h at 110 mC in 6 M HCl. The phosphoamino acids were analysed as described by Kamps & Sefton (1989) using the BAS-1500 system. Results Expression of recombinant MP E. coli transformants containing the expression plasmid pGEX-30K or the control vector plasmid pGEX-5X-2 were induced with IPTG and the recombinant proteins were affinitypurified on a glutathione–Sepharose column. The identity of the recombinant 56 kDa MP protein (GST–MP) was confirmed by Western blot analysis using anti-GST (Amersham Pharmacia) and anti-MP antibodies (Meshi et al., 1992) with GST and histidine-tagged recombinant MP from pLQV5 plasmid (Meshi et al., 1992) serving as negative and positive controls, respectively (data not shown). GST–MP was mostly soluble and was used in an RNA-binding assay on nitrocellulose membrane. GST–MP and single-stranded DNA binding protein used as a positive control showed dosedependent RNA-binding activity, while GST and BSA used as negative controls showed no such activity (data not shown). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 Protein kinase complex with ToMV MP Fig. 1. Protein-complex kinase assay with GST–MP. Aliquots of glutathione–Sepharose beads conjugated with GST (lanes 1–3) or GST–MP (lanes 4–6) were incubated with PBS (lanes 1 and 4), an extract of tobacco leaves (lanes 2 and 5) or BY-2 cells (lanes 3 and 6) and used for phosphorylation reactions after washing the beads with NETN buffer. The reaction products were subjected to SDS–PAGE through a 10 % gel and visualized by Coomassie blue staining (a). The same gel was analysed by autoradiography (b). Arrows indicate the positions of the protein bands corresponding to GST and GST–MP. Positions of molecular mass (kDa) markers are shown on the left. Fig. 2. Effect of salt concentration on the MP–kinase complex. Aliquots of glutathione–Sepharose beads conjugated with GST–MP were incubated with BY-2 cell extract and washed with NETN buffer containing 0n15 (lane 1), 0n30 (lane 2), 0n60 (lane 3), 1n5 (lane 4) or 3n0 (lane 5) M NaCl. After subsequent washing with the kinase buffer, the beads were used for phosphorylation reactions and analysed as in Fig. 1. The arrow in the autoradiogram indicates GST–MP. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of GST–MP detected by staining with Coomassie blue. Fig. 3. Depletion of protein kinase activity in the cell extract by pull-down with GST–MP immobilized on beads. BY-2 cell extract was preincubated with glutathione–Sepharose beads conjugated with no protein (lane 2), GST (lane 3) or GST–MP (lane 4). After centrifugation, the supernatant fractions (lanes 2–4) separated from the beads were subjected to the protein-complex kinase assay using fresh batches of GST–MP beads as in Fig. 1. Extract without preincubation was used as a control (lane 1). The arrow in the autoradiogram indicates GST–MP. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of GST–MP detected by staining with Coomassie blue. Protein-complex kinase assay with plant extracts Preliminary experiments showed that GST–MP immobilized on glutathione–Sepharose beads was phosphorylated in vitro by protein kinase activities in the crude extracts of leaves of N. tabacum and BY-2 cells. To avoid effects of proteases and protein phosphatases possibly present in the extracts, we developed a protein-complex kinase assay in which GST–MP was immobilized on the beads, incubated with plant cell extract, and washed thoroughly with NETN buffer before incubation with [γ-$#P]ATP. During the incubation with Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 CAJH Y. Matsushita and others Fig. 4. Effects of protein kinase inhibitors on the phosphorylation of GST–MP. Protein-complex kinase assay was carried out using BY-2 cell extract and GST–MP as in Fig. 1. The assay was performed in the absence (lane 1) or presence (other lanes) of various amounts of inhibitors. The identity and the concentration of each inhibitor were as follows : (a) heparin, 0n1 (lane 2), 1 (lane 3), 10 µg/ml (lane 4) ; (b) suramin, 5 (lane 2), 10 (lane 3), 50 µM (lane 4) ; (c) quercetin, 10 (lane 2), 100 (lane 3), 1000 µM (lane 4) ; (d ) GF109203X, 0n1 (lane 2), 10 µM (lane 3) ; (e) H-89, 1 (lane 2), 10 (lane 3), 100 µM (lane 4) ; ( f ) KN-62, 0n1 (lane 2), 1 (lane 3), 10 µM (lane 4) ; (g) genistein, 1 (lane 2), 10 µM (lane 3). Arrows in the autoradiograms indicate GST–MP. Positions of molecular mass (kDa) markers are shown on the left. The lower panels show the amount of GST–MP after staining with Coomassie blue. plant cell extract at 4 mC, GST–MP could form a stable protein complex with a protein kinase or kinases in the extract. As shown in Fig. 1, GST–MP was phosphorylated by such a kinase activity present in both the cell extracts from tobacco leaves (lane 5) and BY-2 (lane 6), while GST was not phosphorylated by either cell extract (lanes 2 and 3). As shown in Fig. 2, phosphorylation of GST–MP was observed even after washing the beads with NETN buffer containing 3n0 M NaCl. To exclude the possibility that the kinase in the cell extracts formed a complex with the GST moiety of the recombinant protein, a pull-down experiment was carried out. As shown in Fig. 3, the cell extracts were preincubated with the beads conjugated with no protein (lane 2), with GST (lane 3) or with GST–MP (lane 4) before incubation for the protein-complex kinase assay. Preincubation of the cell extracts with GST–MP beads (lane 4) resulted in a decrease in phosphorylation of GST–MP in the assay, while preincubation with beads alone (lane 2) or GST beads (lane 3) had no effect on the phosphorylation compared to a control subjected to no CAJI preincubation (lane 1). The result indicates that the kinase in the cell extracts was pulled down by GST–MP beads through interaction with the MP moiety of GST–MP during the preincubation. Characterization of the protein kinase To determine the type of protein kinase responsible for the phosphorylation of GST–MP, we examined the effects of various protein kinase inhibitors. By using the protein-complex kinase assay, we found that addition of heparin, suramin or quercetin effectively inhibited phosphorylation (Fig. 4 a–c) at concentrations at which these inhibitors are known to inhibit CK II (Hathaway et al., 1980 ; Aboagye-Kwarteng et al., 1991 ; Ruzzene et al., 1992). In contrast, there was no effect on the phosphorylation with other inhibitors such as GF109203X, H-89, KN-62 and genistein, which are known to inhibit protein kinase C, protein kinase A, Ca#+\calmodulin-dependent protein kinase II and tyrosine protein kinase, respectively (Fig. 4 d–g). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 Protein kinase complex with ToMV MP protein-complex kinase assay was diminished in a dosedependent manner by the addition of unlabelled GTP, suggesting that utilization of ATP was competitively inhibited by GTP. Direct evidence for utilization of GTP was provided by incorporation of $#P to MP in the same assay system but with [γ-$#P]GTP as source, a reaction which was also sensitive to heparin (Fig. 5 b). Phosphorylation of mutant MPs Fig. 5. Phosphorylation of GST–MP with GTP. The protein-complex kinase assay was carried out using BY-2 cell extract and GST–MP as in Fig. 1. (a) Assay performed with 22 nM [γ-32P]ATP in the absence (lane 1) or presence of 1 (lane 2) or 10 µM (lane 3) unlabelled GTP. (b) Assay performed in the presence of 110 nM [γ-32P]GTP (34 TBq/mmol) without (lane 1) or with (lane 2) addition of 10 µg/ml heparin. Arrows indicate GST–MP. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of GST–MP after staining with Coomassie blue. Utilization of both GTP and ATP as phosphoryl donor is known to be a unique feature of CK II (Hathaway & Traugh, 1983). As shown in Fig. 5 (a), incorporation of $#P to MP in the Kawakami et al. (1999) reported that in vivo phosphorylation of ToMV MP required serine residues at 37 and 238. Because these residues are in a consensus motif for phosphorylation by CK II, we expressed recombinant MP (GST–MPAA) with alanine residues substituted for serine-37 and serine-238 and tested phosphorylation in our proteincomplex kinase assay. As shown in Fig. 6 (a), there was no difference in protein phosphorylation levels between the wildtype (GST–MPSS) and the mutant (GST–MPAA). Phosphoamino acids were analysed with acid hydrolysate of GST–MPSS and GST–MPAA that had been phosphorylated with [γ-$#P]ATP in the protein-complex kinase assay (Fig. 6 b). Autoradiography after thin-layer electrophoresis indicated that $#P was incorporated into spots corresponding to phosphoserine and phosphothreonine but not phosphotyrosine, which suggests the involvement of a serine\ threonine protein kinase. We next tried to locate the region of phosphorylation by creating truncated recombinant MPs with deletions in the serine-rich C-terminal region. As shown in Fig. 7, phosphorylation was almost completely inhibited for GST– MPdA and GST–MPdSX, in which 9 and 31 amino acids, respectively, were removed from the C termini. This result Fig. 6. Phosphorylation of the recombinant MP with amino acid substitutions. (a) GST–MPSS (wild-type, lane 1) and GST–MPAA (mutant, lane 2) were subjected to the protein-complex kinase assay using BY-2 cell extract as in Fig. 1. The arrow in the autoradiogram indicates the recombinant proteins. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of the recombinant proteins after staining with Coomassie blue. (b) Phosphoamino acid analysis of the protein bands indicated by the arrow in (a). GST–MPSS (lane 1) and GST–MPAA (lane 2) were hydrolysed in HCl and analysed as described in Methods. Positions of phosphoserine, phosphothreonine and phosphotyrosine standards are shown. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 CAJJ Y. Matsushita and others Fig. 7. Phosphorylation of recombinant MPs with C-terminal deletions. GST–MP (lane 1), GST–MPdA (lane 2) and GST–MPdSX (lanes 3) were subjected to the protein-complex kinase assay using BY-2 cell extract as in Fig. 1. Asterisks show the positions of the recombinant proteins. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of the recombinant proteins after staining with Coomassie blue. suggests that the phosphorylation sites are located within the C-terminal 9 amino acids, although we cannot strictly rule out the possibility that these deletions and\or the extra nonviral residues appended to the C terminus caused conformational changes of the recombinant protein leading to loss of affinity to the protein kinase. Discussion Kawakami et al. (1999) carried out in vivo phosphorylation analyses and created a series of mutant ToMV MPs by introducing single or double amino acid replacements. The mutant viral RNAs were inoculated into BY-2 protoplasts to detect $#P-labelled ToMV MP. By comparing $#P incorporation between the wild-type and mutant MPs, they established that serine-37 and serine-238 were the amino acid residues phosphorylated in vivo. Further experiments illustrated an essential role for serine-37 in the function and stability of ToMV MP. It has been reported that CK II of animals and yeast phosphorylates serine and threonine residues within the consensus motif (S\T)XX(D\E) (Pearson & Kemp, 1991). In ToMV MP, there are seven serine\threonine residues placed in such a context, including serine-37 (SKVD) and serine-238 (SFDE) which were phosphorylated as described above. To characterize the cellular kinases responsible for phosphorylation of ToMV MP we took advantage of an in vitro assay system using recombinant MP as a substrate, so that CBAA effects of kinase inhibitors and enzyme–substrate interactions could be assessed directly. The protein-complex kinase assay system employed in this study allowed us to eliminate the effects of various cellular factors such as proteases, phosphatases and other kinases that did not associate with the substrate. The kinase associated with ToMV MP was shown to be inhibited by heparin, suramin and quercetin at concentrations that are reported to inhibit CK II. In contrast, the kinase activity was not affected by other types of protein kinase inhibitor. Atabekov & Taliansky (1990) suggested the possible involvement of cyclic AMP-dependent protein kinase in the phosphorylation of TMV MP. In our in vitro assay system, however, addition of an inhibitor for protein kinase A (H-89) did not affect phosphorylation of GST–MP by BY-2 cell extract. In addition, GST–MP could not serve as a substrate for the catalytic subunit of murine protein kinase A in our simple kinase assay (data not shown). Although protein kinase A has not been reported in higher plants, our results suggest that such an enzyme, if it exists in plants, does not participate directly in the phosphorylation of ToMV MP. In contrast to the in vivo phosphorylation data (Kawakami et al., 1999), the phosphoamino acid analysis reported here indicated that both serine and threonine residues were phosphorylated in the protein-complex kinase assay. Furthermore, the levels of phosphorylation at serine and threonine residues of the mutant GST–MPAA were comparable to the wild-type. These results suggest that some mechanism may exist whereby the in vivo phosphorylation status of MP is strictly controlled. Perhaps the structure of MP may be sensitive in vivo to some protein modification other than phosphorylation, which results in steric hindrance and inaccessibility to the cellular protein kinases. It is also possible that the CK II-like protein kinase activity detected in our study may be different from the one responsible for the phosphorylation of serine-37 and\or serine-238 (Kawakami et al., 1999). However, our in vitro study, focusing on the particular kinase that formed a stable complex with the substrate, need not necessarily be in contradiction with the results of the in vivo study, which could reflect a steady-state level of phosphorylation of the substrate as it is interacting with various cellular factors such as phosphatases and possible endogenous kinase inhibitors. Perhaps multiple protein kinases distributed in different cellular compartments may participate in the phosphorylation of ToMV MP. In fact, our simple kinase assay with cellular extracts resulted in a significant level of phosphorylation of GST–MP that could not be diminished by the CK II inhibitors (data not shown). Citovsky et al. (1993) have detected a cell wall-associated protein kinase involved in the phosphorylation of serine-258, threonine-261 and serine-265 of TMV MP. Although the MP of this TMV strain has a somewhat different C-terminal sequence from that of ToMV, threonine-261 and serine-265 of TMV MP may correspond to serine-257 and serine-261 of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 Protein kinase complex with ToMV MP Breeding ’ from the Ministry of Agriculture, Forestry and Fisheries of Japan (to H. N.). References Aboagye-Kwarteng, T., Ole-Moiyoi, O. K. & Lonsdale-Eccles, J. D. (1991). Phosphorylation differences among proteins of bloodstream Fig. 8. C-terminal serine/threonine clusters in MPs of two viruses. A part of the amino acid sequence of ToMV MP (Ohno et al., 1984) and of TMV MP (Goelet et al., 1982) are shown in one-letter code and aligned. Asterisks indicate matched residues. Arrows indicate the positions of C-terminal truncations in GST–MPdSX (downstream of proline-234) and GST–MPdA (downstream of threonine-256). ToMV MP, respectively (Fig. 8). Our in vitro assay using the Cterminally truncated GST–MPs indicated that deletions of these residues (in addition to threonine-256 of ToMV MP) resulted in almost complete loss of phosphorylation. Since the kinase assay is dependent on complex formation between GST–MP and cellular protein(s), the loss of phosphorylation may be attributable either to the absence of the target residues for the protein kinase or failure of the complex to form due to a conformational change in the substrate. In either case, it should be noted that we used a buffer without any detergents to prepare cell extracts, which hence should contain only soluble material and may not contain cell-wall associated proteins. According to Citovsky et al. (1993), the cell wallassociated kinase was absent from the soluble fraction. Thus, the CK II-like protein kinase in our study would be distinct from the cell wall-associated kinase reported by Citovsky et al. (1993). Padgett et al. (1996) reported a dynamic aspect of the cellular distribution of MP, which varied spatiotemporally from the early to the late stages of infection. MP probably interacts with various cellular components to manifest its multiple functions at various subcellular locations including the cortical ER, microtubules and plasmodesmata. Thus there may be several protein kinases that can phosphorylate MP so as to regulate its interaction with various cellular proteins. It is not known whether the CK II-like protein kinase described here binds directly to MP or associates with MP through a tethering protein. Further work will be required for the determination of the specific phosphorylation sites and the identification of the protein kinase, information which should help understand the significance of the complex formation between the protein kinase and MP in infected cells. We are indebted to Dr Yoshimi Okada for general guidance and valuable suggestions. We thank Dr Hideki Takahashi and Dr Toshiyuki Nagata for providing a tobacco strain and BY-2 cell line, respectively. This work was supported by the Grant-in-Aid for Encouragement of Young Scientists from Ministry of Education, Science, Sports and Culture of Japan (to Y. M., no. 11760033) and the Grant-in-Aid ‘ Integrated Research Program for the Use of Biotechnological Procedures for Plant developmental stages of Trypanosoma brucei brucei. Biochemical Journal 275, 7–14. Atabekov, J. G. & Dorokhov, Y. L. (1984). Plant virus-specific transport function and resistance of plants to viruses. Advances in Virus Research 29, 313–364. Atabekov, J. G. & Taliansky, M. E. (1990). Expression of a plant viruscoded transport function by different viral genomes. Advances in Virus Research 38, 201–248. Atkins, D., Hull, R., Wells, B., Roberts, K., Moore, P. & Beachy, R. N. (1991 a). The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localized to plasmodesmata. Journal of General Virology 72, 209–211. Atkins, D., Roberts, K., Hull, R., Prehaud, C. & Bishop, D. H. (1991 b). Expression of the tobacco mosaic virus movement protein using a baculovirus expression vector. Journal of General Virology 72, 2831–2835. Carrington, J. C., Kasschau, K. D., Mahajan, S. K. & Schaad, M. C. (1996). Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 8, 1669–1681. Citovsky, V., Knorr, D., Schuster, G. & Zambryski, P. (1990). The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60, 637–647. Citovsky, V., McLean, B. G., Zupan, J. R. & Zambryski, P. (1993). Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase. Genes & Development 7, 904–910. Deom, C. M., Oliver, M. J. & Beachy, R. N. (1987). The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement. Science 237, 389–394. Deom, C. M., Lapidot, M. & Beachy, R. N. (1992). Plant virus movement proteins. Cell 69, 221–224. Fenczik, C. A., Padgett, H. S., Holt, C. A., Casper, S. J. & Beachy, R. N. (1995). Mutational analysis of the movement protein of odontoglossum ringspot virus to identify a host-range determinant. Molecular Plant– Microbe Interactions 8, 666–673. Fujita, M., Mise, K., Kajiura, Y., Dohi, K. & Furusawa, I. (1998). Nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus. Journal of General Virology 79, 1273–1280. Goelet, P., Lomonossoff, G. P., Butler, P. J., Akam, M. E., Gait, M. J. & Karn, J. (1982). Nucleotide sequence of tobacco mosaic virus RNA. Proceedings of the National Academy of Sciences, USA 79, 5818–5822. Haley, A., Hunter, T., Kiberstis, P. & Zimmern, D. (1995). Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco protoplasts. Plant Journal 8, 715–724. Hathaway, G. M. & Traugh, J. A. (1983). Casein kinase II. Methods in Enzymology 99, 317–331. Hathaway, G. M., Lubben, T. H. & Traugh, J. A. (1980). Inhibition of casein kinase II by heparin. Journal of Biological Chemistry 255, 8038–8041. Kaelin, W. G., Jr, Pallas, D. C., DeCaprio, J. A., Kaye, F. J. & Livingston, D. M. (1991). Identification of cellular proteins that can interact specifically with the T\E1A-binding region of the retinoblastoma gene product. Cell 64, 521–532. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25 CBAB Y. Matsushita and others Kamps, M. A. & Sefton, B. M. (1989). Acid and base hydrolysis of phospho-proteins bound to Immobilon : a rapid technique for the analysis of phosphoamino acids in gel-fractionated proteins. Analytical Biochemistry 176, 22–27. Kawakami, S., Padgett, H. S., Hosokawa, D., Okada, Y., Beachy, R. N. & Watanabe, Y. (1999). Phosphorylation and\or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo. Journal of Virology 73, 6831–6840. Li, Q. & Palukaitis, P. (1996). Comparison of the nucleic acid- and NTPbinding of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. Virology 216, 71–79. Lucas, W. J. & Gilbertson, R. L. (1994). Plasmodesmata in relation to viral movement within leaf tissues. Annual Review of Phytopathology 32, 387–411. Meshi, T., Watanabe, Y., Saito, T., Sugimoto, A., Maeda, T. & Okada, Y. (1987). Function of the 30 kd protein of tobacco mosaic virus : involvement in cell-to-cell movement and dispensability for replication. EMBO Journal 6, 2557–2563. Meshi, T., Motoyoshi, F., Maeda, T., Yoshiwoka, S., Watanabe, H. & Okada, Y. (1989). Mutations in the tobacco mosaic virus 30-kD protein gene overcome Tm-2 resistance in tomato. Plant Cell 1, 515–522. Meshi, T., Hosokawa, D., Kawagishi, M., Watanabe, Y. & Okada, Y. (1992). Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA. Virology 187, 809–813. Mise, K., Allison, R. F., Janda, M. & Ahlquist, P. (1993). Bromovirus movement protein genes play a crucial role in host specificity. Journal of Virology 67, 2815–2823. Nagata, T., Okada, K., Takebe, I. & Matsui, C. (1981). Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reversephase evaporation vesicles (liposomes). Molecular and General Genetics 184, 161–165. Ohno, T., Aoyagi, M., Yamanashi, Y., Saito, H., Ikawa, S., Meshi, T. & Okada, Y. (1984). Nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome. Journal of Biochemistry (Tokyo) 96, 1915–1923. CBAC Padgett, H. S., Epel, B. L., Kahn, T. W., Heinlein, M., Watanabe, Y. & Beachy, R. N. (1996). Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection. Plant Journal 10, 1079–1088. Pearson, R. B. & Kemp, B. E. (1991). Protein kinase phosphorylation site sequences and consensus specificity motifs : tabulations. Methods in Enzymology 200, 62–81. Reichel, C., Mas, P. & Beachy, R. N. (1999). The role of the ER and cytoskeleton in plant viral trafficking. Trends in Plant Science 4, 458–462. Ruzzene, M., Vianello, F., Donella-Deana, A. & Deana, R. (1992). Purification and characterization of two casein kinases from ejaculated bovine spermatozoa. Journal of Biochemistry (Tokyo) 112, 768–774. Taliansky, M. E., Malyshenko, S. I., Pshennikova, E. S. & Atabekov, J. G. (1982). Plant virus-specific transport function II : a factor controlling virus host range. Virology 122, 327–331. Tomenius, K., Clapham, D. & Meshi, T. (1987). Localization by immunogold cytochemistry of the virus-coded 30K protein in plasmodesmata of leaves infected with tobacco mosaic virus. Virology 160, 363–371. Watanabe, Y., Emori, Y., Ooshika, I., Meshi, T., Ohno, T. & Okada, Y. (1984). Synthesis of TMV-specific RNAs and proteins at the early stage of infection in tobacco protoplasts : transient expression of the 30K protein and its mRNA. Virology 133, 18–24. Watanabe, Y., Ogawa, T. & Okada, Y. (1992). In vivo phosphorylation of the 30-kDa protein of tobacco mosaic virus. FEBS Letters 313, 181–184. Weber, H. & Pfitzner, A. J. (1998). Tm-2(2) resistance in tomato requires recognition of the carboxy terminus of the movement protein of tomato mosaic virus. Molecular Plant–Microbe Interactions 11, 498–503. Weber, H., Schultze, S. & Pfitzner, A. J. (1993). Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome the Tm-2(2) resistance gene in the tomato. Journal of Virology 67, 6432–6438. Received 15 February 2000 ; Accepted 25 April 2000 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 11 May 2017 13:24:25