Download In vitro phosphorylation of the movement protein of tomato mosaic

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of General Virology (2000), 81, 2095–2102. Printed in Great Britain
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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
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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).
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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
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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).
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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.
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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
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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
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(1991 a). The tobacco mosaic virus 30K movement protein in transgenic
tobacco plants is localized to plasmodesmata. Journal of General Virology
72, 209–211.
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Received 15 February 2000 ; Accepted 25 April 2000
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