Gene Research Center, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan1
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan2
National Institute of Agrobiological Resources, 2-1-2 Kan-nondai, Tsukuba, Ibaraki 305-8602, Japan3
Author for correspondence: Hiroshi Nyunoya. Fax +81 42 367 5563. e-mail nyunoya{at}cc.tuat.ac.jp
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Abstract |
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Introduction |
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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.
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Methods |
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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 [-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 [-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 [-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.
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Results |
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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 [-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.
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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.
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Discussion |
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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)
.
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Acknowledgments |
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References |
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Received 15 February 2000;
accepted 25 April 2000.