In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase

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., 1991a ). MPs are also reported to be involved in host 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. (1991b ) showed that plant-expressed TMV MP comigrates during SDSPAGE 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 RNA-inoculated 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 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 host-plant kinases, we prepared a recombinant MP and established a protein-complex kinase assay system.

Plasmid construction.
The 1·0 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 3' termini and inserted into the SmaI site of pBluescriptII SK(+) (Stratagene) to create pBS-30K, with the coding sequence in the EcoRI to NotI sense of the vector. The 1·0 kb EcoRINotI fragment was subsequently inserted into the EcoRINotI sites of pGEX-5X-2 (Amersham Pharmacia) to produce pGEX-30K, which encodes glutathione S-transferase (GST)-fused ToMV MP.

The 1·0 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 3' termini and inserted into the SmaI site of pGEX-6P-3 (Amersham Pharmacia) to create pGEX-30KSS and pGEX-30KS37AS238A, 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 0·54 kb AatII or 0·31 kb StuIXbaI 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 GSTMP; pGEX-30KSS for GSTMPSS; pGEX-30KS37AS238A for GSTMPAA; pGEX-30KdA for GSTMPdA; pGEX-30KdSX for GSTMPdSX. The recombinant protein GSTMPdA had the C-terminal 9 amino acids replaced by 27 nonviral residues (QVALFGEMCAEPLFVYFSKYIQICIRS) derived from the vector. Another recombinant protein, GSTMPdSX, had the C-terminal 31 amino acids replaced by 7 residues (LERPHRD) derived from the vector. Protein expression was induced by addition of 0·2 mM IPTG. The recombinant proteins were purified using glutathioneSepharose 4B beads (Amersham Pharmacia) as described by Kaelin et al. (1991) and stored in a modified NETN buffer (50 mM TrisHCl, pH 8·0, 1 mM EDTA, 150 mM NaCl, 0·5% 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 (8 h) cycle at 24 °C. Suspension cultures of a BY-2 tobacco cell line were maintained as described by Nagata et al. (1981) and cells in the late-exponential phase were frozen at -80 °C after washing with PBS (137 mM NaCl, 2·68 mM KCl, 10·1 mM Na₂HPO₄, 1·76 mM KH₂PO₄, pH 7·4). To prepare the cell extracts, leaves (0·1 g fresh wt/ml) of the tobacco plants and frozen BY-2 cells (0·4 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 16000 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, glutathioneSepharose 4B beads conjugated to 1 µg of recombinant protein were suspended in 100 µl of kinase buffer (40 mM HEPES, pH 7·4, 10 mM MgCl₂, 3 mM MnCl₂) including 45 µl cell extract as prepared above plus the protease inhibitors pepstatin A (1 µg/ml), aprotinin (2 µg/ml), chymostatin (0·1 µg/ml), leupeptin (0·5 µg/ml) and trans-epoxysuccinyl-L-leucylamido-[4-guanidino]butane (7·2 µg/ml). The phosphorylation assay was started by addition of 370 kBq [γ-³²P]ATP (168 TBq/mmol), incubated for 30 min at 25 °C on a rotator, and terminated by washing the beads twice with 0·9 ml NETN buffer.

For the protein-complex kinase assay, glutathioneSepharose 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 °C 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-³²P]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 GSTMP. Protein phosphorylation was analysed by SDSPAGE followed by image analysis with a BAS-1500 system (Fuji Photo Film).

Phosphoamino acid analysis.
Proteins phosphorylated with [γ-³²P]ATP were separated by SDSPAGE and blotted onto PVDF membrane. The protein band was excised and hydrolysed for 2 h at 110 °C in 6 M HCl. The phosphoamino acids were analysed as described by Kamps & Sefton (1989) using the BAS-1500 system.

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 affinity-purified on a glutathioneSepharose column. The identity of the recombinant 56 kDa MP protein (GSTMP) 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). GSTMP was mostly soluble and was used in an RNA-binding assay on nitrocellulose membrane. GSTMP and single-stranded DNA binding protein used as a positive control showed dose-dependent RNA-binding activity, while GST and BSA used as negative controls showed no such activity (data not shown).

Protein-complex kinase assay with plant extracts
Preliminary experiments showed that GSTMP immobilized on glutathioneSepharose 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 GSTMP was immobilized on the beads, incubated with plant cell extract, and washed thoroughly with NETN buffer before incubation with [γ-³²P]ATP. During the incubation with plant cell extract at 4 °C, GSTMP could form a stable protein complex with a protein kinase or kinases in the extract. As shown in Fig. 1, GSTMP 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 GSTMP was observed even after washing the beads with NETN buffer containing 3·0 M NaCl.

(32K): Fig. 1. Protein-complex kinase assay with GSTMP. Aliquots of glutathioneSepharose beads conjugated with GST (lanes 13) or GSTMP (lanes 46) 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 SDSPAGE 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 GSTMP. Positions of molecular mass (kDa) markers are shown on the left.

(90K): Fig. 2. Effect of salt concentration on the MPkinase complex. Aliquots of glutathioneSepharose beads conjugated with GSTMP were incubated with BY-2 cell extract and washed with NETN buffer containing 0·15 (lane 1), 0·30 (lane 2), 0·60 (lane 3), 1·5 (lane 4) or 3·0 (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 GSTMP. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of GSTMP detected by staining with Coomassie blue.

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 GSTMP (lane 4) before incubation for the protein-complex kinase assay. Preincubation of the cell extracts with GSTMP beads (lane 4) resulted in a decrease in phosphorylation of GSTMP 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 preincubation (lane 1). The result indicates that the kinase in the cell extracts was pulled down by GSTMP beads through interaction with the MP moiety of GSTMP during the preincubation.

(55K): Fig. 3. Depletion of protein kinase activity in the cell extract by pull-down with GSTMP immobilized on beads. BY-2 cell extract was preincubated with glutathioneSepharose beads conjugated with no protein (lane 2), GST (lane 3) or GSTMP (lane 4). After centrifugation, the supernatant fractions (lanes 24) separated from the beads were subjected to the protein-complex kinase assay using fresh batches of GSTMP beads as in Fig. 1. Extract without preincubation was used as a control (lane 1). The arrow in the autoradiogram indicates GSTMP. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of GSTMP detected by staining with Coomassie blue.

Characterization of the protein kinase
To determine the type of protein kinase responsible for the phosphorylation of GSTMP, 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. 4ac) 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, Ca²⁺/calmodulin-dependent protein kinase II and tyrosine protein kinase, respectively (Fig. 4dg).

(52K): Fig. 4. Effects of protein kinase inhibitors on the phosphorylation of GSTMP. Protein-complex kinase assay was carried out using BY-2 cell extract and GSTMP 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, 0·1 (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, 0·1 (lane 2), 10 µM (lane 3); (e) H-89, 1 (lane 2), 10 (lane 3), 100 µM (lane 4); (f) KN-62, 0·1 (lane 2), 1 (lane 3), 10 µM (lane 4); (g) genistein, 1 (lane 2), 10 µM (lane 3). Arrows in the autoradiograms indicate GSTMP. Positions of molecular mass (kDa) markers are shown on the left. The lower panels show the amount of GSTMP 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 ³²P to MP in the protein-complex kinase assay was diminished in a dose-dependent 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 ³²P to MP in the same assay system but with [γ-³²P]GTP as source, a reaction which was also sensitive to heparin (Fig. 5b).

(57K): Fig. 5. Phosphorylation of GSTMP with GTP. The protein-complex kinase assay was carried out using BY-2 cell extract and GSTMP 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 GSTMP. Positions of molecular mass (kDa) markers are shown on the left. The lower panel shows the amount of GSTMP after staining with Coomassie blue.

Phosphorylation of mutant MPs
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 (GSTMPAA) with alanine residues substituted for serine-37 and serine-238 and tested phosphorylation in our protein-complex kinase assay. As shown in Fig. 6(a), there was no difference in protein phosphorylation levels between the wild-type (GSTMPSS) and the mutant (GSTMPAA).

(37K): Fig. 6. Phosphorylation of the recombinant MP with amino acid substitutions. (a) GSTMPSS (wild-type, lane 1) and GSTMPAA (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). GSTMPSS (lane 1) and GSTMPAA (lane 2) were hydrolysed in HCl and analysed as described in Methods. Positions of phosphoserine, phosphothreonine and phosphotyrosine standards are shown.

Phosphoamino acids were analysed with acid hydrolysate of GSTMPSS and GSTMPAA that had been phosphorylated with [γ-³²P]ATP in the protein-complex kinase assay (Fig. 6b). Autoradiography after thin-layer electrophoresis indicated that ³²P 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 GSTMPdA and GSTMPdSX, in which 9 and 31 amino acids, respectively, were removed from the C termini. This result 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.

(69K): Fig. 7. Phosphorylation of recombinant MPs with C-terminal deletions. GSTMP (lane 1), GSTMPdA (lane 2) and GSTMPdSX (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.

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 ³²P-labelled ToMV MP. By comparing ³²P 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 effects of kinase inhibitors and enzymesubstrate 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 GSTMP by BY-2 cell extract. In addition, GSTMP 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 GSTMPAA 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 GSTMP 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 ToMV MP, respectively (Fig. 8). Our in vitro assay using the C-terminally truncated GSTMPs 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 GSTMP 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 wall-associated 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) .

(16K): 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 GSTMPdSX (downstream of proline-234) and GSTMPdA (downstream of threonine-256).

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 Breeding from the Ministry of Agriculture, Forestry and Fisheries of Japan (to H.N.).