From the Institute of Biotechnology, Viikki Biocenter, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland
Received for publication, October 19, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Plant viruses encode movement proteins (MPs) to
facilitate transport of their genomes from infected into neighboring
healthy cells through plasmodesmata. Growing evidence suggests that
specific phosphorylation events can regulate MP functions. The coat
protein (CP) of potato virus A (PVA; genus Potyvirus) is a
multifunctional protein involved both in virion assembly and virus
movement. Labeling of PVA-infected tobacco leaves with
[33P]orthophosphate demonstrated that PVA CP is
phosphorylated in vivo. Competition assays established that
PVA CP and the well characterized 30-kDa MP of tobacco mosaic
virus (genus Tobamovirus) are phosphorylated
in vitro by the same Ser/Thr kinase activity from tobacco
leaves. This activity exhibits a strong preference for Mn2+
over Mg2+, can be inhibited by micromolar concentrations of
Zn2+ and Cd2+, and is not
Ca2+-dependent. Tryptic phosphopeptide mapping
revealed that PVA CP was phosphorylated by this protein kinase activity
on multiple sites. In contrast, PVA CP was not phosphorylated when
packaged into virions, suggesting that the phosphorylation sites are
located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that
protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in
planta.
A delicate balance between protein phosphorylation and
dephosphorylation regulates the function of a vast variety of proteins in the cell. Recently, several lines of evidence have suggested that
phosphorylation of plant virus-encoded movement proteins (MPs)1 by host plant protein
kinases may be involved in the process of virus movement (1-3). The
functional role of MPs is to assist the spread of viral progeny from
cell to cell and over long distances (reviewed in Refs. 4-7). There is
evidence that the 30-kDa MP of tobacco mosaic virus (TMV; genus
Tobamovirus) is phosphorylated when expressed in insect
cells from a baculovirus vector (8), in TMV-infected protoplasts (9,
10), and in the cell wall-enriched fractions of transgenic plants
expressing the wild-type MP and its mutants (3, 11). The 17-kDa MP of
potato leafroll virus (genus Luteovirus) was shown to be
phosphorylated in a reconstituted system containing bacterially
expressed protein and membrane preparations from potato leaves (12). In
another report, phosphorylation of the 69-kDa MP of turnip yellow
mosaic virus (genus Tymovirus) was demonstrated when the MP
gene was expressed in insect cells using a baculovirus vector (13).
It is not yet clear whether MP phosphorylation is essential for the
general process of virus movement; however, there is growing evidence
suggesting that phosphorylation can affect several MP functions.
Originally, it was proposed that phosphorylation represents a mechanism
for MP inactivation and sequestration in the cell walls of mature
plants (11). Proteolytic processing was found to be an alternative
mechanism to phosphorylation for inactivation of TMV MP in
Arabidopsis thaliana (14). Recently, new evidence has
accumulated that suggests that phosphorylation of TMV MP may directly
affect its function. Either phosphorylation or the presence of serine
37 in MP of tomato mosaic virus (genus Tobamovirus) was
shown to be essential for the protein intracellular localization and
stability and, therefore, required for the efficient spread of the
virus (1). These results indicated that phosphorylation of MPs by
cellular protein kinase(s) may represent an active process required by
the plant viruses to execute their movement function. Second line of
evidence in support of the possible involvement of MP phosphorylation
in the cell-to-cell movement came from in vitro studies
showing that the phosphorylation of TMV MP abolishes its ability to
repress RNA translation (2). This finding suggested a possible
mechanism for how MP phosphorylation may regulate the function of
movement ribonucleoprotein intermediates in the course of their
cell-to-cell translocation. According to this hypothesis, MP
phosphorylation converts the translation-incompetent movement intermediates into the translation-ready state, thus allowing the virus
to replicate in the newly infected cell. In another recent study (3),
TMV MP mutant mimicking phosphorylation was reported to be deficient in
plasmodesmal transport, suggesting that phosphorylation is involved in
down-regulation of the MP activity.
Although much is already known about the phosphorylation of MPs of
tobamoviruses, data on the phosphorylation of potyvirus proteins
involved in movement are completely missing. In contrast to
tobamoviruses, potyviruses do not encode a particular dedicated movement protein, using instead several polyfunctional proteins to
execute their movement function. These proteins, designated movement-related proteins (MRPs), include the coat protein (CP), the
helper component protease (HC-Pro), the cylindrical inclusion protein
(CIP), and the genome-linked protein (VPg) (reviewed in Ref. 15). In
this study, we report that two out of the four MRPs of potato virus A
(PVA; genus Potyvirus), CP and VPg, are phosphorylated by
protein kinases from Nicotiana tabacum. Our further results
suggest that phosphorylation of both PVA CP and TMV MP involves the
same Ser/Thr-specific plant protein kinase activity. Finally, we
show that phosphorylation of PVA CP by this enzymatic activity affects
its ability to bind RNA. This suggests a strategy for how the formation
and/or stability of viral ribonucleoproteins is regulated in
planta.
Virus Infection, Purification, and Coat Protein
Extraction--
PVA strain B11 was propagated in N. tabacum
(cv. SR1) plants. To establish systemic infection, tobacco plants were
mechanically inoculated by grinding PVA-infected leaves at 1 g per
4 ml of distilled water and rubbing the sap into the lower leaves using carborundum as an abrasive. PVA infection was monitored by
immunoblotting with mouse anti-PVA antibody (Bioreba). The virus was
purified using the method described in Ref. 16, and the resulting virus preparations were dialyzed against 25 mM HEPES, pH 7.4. The
coat protein was extracted from virus particles by guanidine HCl and LiCl methods as described in (17) and dialyzed against the same HEPES
buffer containing 0.5 M NaCl.
Plant Material and Preparation of Plant Protein
Extracts--
Tobacco plants (N. tabacum cv.
SR1) were grown under nonsterile conditions in controlled
environmental chambers using soil mixed with vermiculite (1:2). All
plants were cultivated under long day conditions with 16 h of
light (at 23 °C) and 8 h of darkness (at 23 °C). Fully
expanded leaves of 10 cm in length were harvested and used in standard
phosphorylation assays. Leaf tissue was homogenized with pestle and
mortar prechilled to 4 °C for 5 min in homogenization medium
containing 25 mM HEPES, pH 7.4, and 0.25 M
sucrose. A protease inhibitor mixture (Roche Molecular Biochemicals)
was added at concentrations recommended by the manufacturer to
homogenization medium upon commencement of grinding. The ground
extracts were filtered through one layer of Miracloth (Calbiochem) and
used as a kinase source for in vitro assays.
Plasmid Construction--
Generation of expression constructs
was described in Refs. 18 and 19. Briefly, the coding sequences of four
PVA MRPs (CIP, CP, HC-Pro, and VPg) and TMV MP were isolated by
polymerase chain reaction and cloned into pQE-30 or pQE-9 plasmid
vectors (Qiagen), allowing
isopropyl-1-thio- Fusion Protein Purification--
The purification scheme of
bacterially expressed His6PVA MRPs and His6TMV
MP was previously described (18, 19). In the current study, the same
purification strategy was implemented with the following modifications.
Bacterial cell pellets were resuspended in buffer containing 20 mM Tris-HCl, pH 7.4, 0.2 mM NaCl, 1 mM EDTA, 10% sucrose and then lysed in a French press cell
(two cycles at 10,000 p.s.i.). Cell lysates were centrifuged (12,000 × g for 10 min at 4 °C), and the pellets
were solubilized in buffer A (6 M guanidine HCl, 0.1 M
Na2HPO4/NaH2PO4, 0.01 M Tris-HCl, pH 8.0). After incubation for 30 min at room
temperature with agitation, the insoluble material was removed from the
solution by centrifugation (12,000 × g for 10 min at
4 °C). The supernatant was applied to the column containing
Ni2+-NTA-agarose (Qiagen) and chromatographed. His-tagged
proteins were eluted by step changes in pH. The obtained pure proteins were refolded by a rapid dialysis procedure, which was previously successfully applied to recover the RNA binding activity of PVA MRPs
(18) and TMV MP (19).
In Vitro Synthesis of RNA--
In vitro transcription
reactions were performed as described in Ref. 18.
32P-Labeled transcript corresponding to the 5'-untranslated
region of PVA RNA (160 nucleotides in length, positive polarity) was generated using an in vitro transcription system (Riboprobe;
Promega).
SDS-PAGE and Immunoblotting--
Samples were solubilized at
room temperature in 1× SDS-PAGE sample buffer (2% (w/v) SDS, 5%
2-mercaptoethanol, 10% glycerol, 0.05 M Tris-HCl, pH 6.8),
and loaded on 12.5% (w/v) SDS-polyacrylamide gels. The gel
electrophoresis was performed as described in Ref. 20. Following
electrophoresis, gels were stained by Coomassie Brilliant Blue R-250,
or polypeptides were electrophoretically transferred to polyvinylidene
difluoride membranes (Immobilon-P; Millipore Corp.). For Western
analysis, membranes were blocked for 1 h in Tris-buffered saline
containing 0.05% Tween 20 and 1% (w/v) bovine serum albumin (BSA).
Blots were incubated for 2 h at room temperature with
affinity-purified rabbit anti-phosphotyrosine antibody
(Zymed Laboratories Inc.) diluted 1:1000 in 1% (w/v) BSA. Alkaline phosphatase-coupled anti-rabbit IgG (diluted 1:5000) was
used to reveal the presence of the primary antibodies. Prior to RNA
binding or immunoblotting, amounts of protein were normalized by
comparing band intensity on Amido Black- or Ponceau S-stained membranes. Radioactively labeled proteins or RNA-protein complexes were
visualized by autoradiography with Eastman Kodak Co. BMR film or
quantified by using a phosphor imager (Fuji) and Tina 2.09c
software (Raytest).
In Vivo Phosphorylation--
Source leaves of PVA-infected or
mock-infected tobacco plants were cut in disks (1 cm in diameter) and
incubated in 25 mM HEPES, pH 6.8, containing 1 mCi (0.5 mCi
ml In Vitro Kinase Assays--
Phosphorylation was measured as the
incorporation of radioactivity from [ In Vitro Enzymatic Dephosphorylation Assays--
Protein
dephosphorylation was analyzed as the loss of
[33P]phosphate from labeled proteins following their
separation by SDS-PAGE. Two types of enzymes were used in
dephosphorylation assays: general shrimp alkaline phosphatase (SAP; 1 unit/µl; Amersham Pharmacia Biotech) and Ser/Thr-specific protein
phosphatase-2A (PPTase-2A, 0.5 units/µl; Promega). Digestion of
phosphoproteins with SAP was performed at 37 °C for 15 min in a
final volume of 20 µl containing 1 µg of phosphorylated substrate
protein, 3 units of SAP, 10 mM MgCl2, and 20 mM Tris-HCl, pH 8.0. Protein dephosphorylation with PPTase-2A was carried out by incubation of the 20-µl sample
containing 1 µg of phosphorylated substrate protein, 0.5 units of
PPTase-2A, 1 mM MnCl2, 1 mM
dithiothreitol, and 20 mM Tris-HCl, pH 7.5, for 20 min at
37 °C. Control reactions in the same buffer with 1 µM okadaic acid (Calbiochem) were performed. The dephosphorylation reactions were terminated by adding 5 µl of 5× SDS-PAGE sample buffer followed immediately by boiling for 5 min.
Tryptic Phosphopeptide Mapping--
Tryptic phosphopeptide
mapping was carried out as described in Ref. 21.
RNA-Protein Blotting--
The RNA-protein binding assays were
performed according to the method described in Ref. 22 with some
modifications. Equal amounts (1 µg) of target protein were
phosphorylated in vitro in the presence of 500 µM unlabeled ATP and 10 mM Mn2+
as described above. The phosphoproteins were separated from
unincorporated ATP by SDS-PAGE and transferred to Immobilon-P
membranes. The membranes were blocked for 1 h in RNA binding
buffer (20 mM HEPES, 6 mM Tris-HCl, pH 7.0, 25 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) containing 5%
(w/v) nonfat milk powder. After three washes with RNA binding buffer,
SDS was removed from the blotted proteins by guanidine HCl extraction.
For this purpose, membranes were incubated for 10 min in RNA binding
buffer with 6 M guanidine HCl. The proteins were further
renatured by successive incubations of 10 min each in RNA binding
buffer containing gradually decreasing concentrations of guanidine HCl
(3, 1.5, 0.75, 0.38, 0.19, and 0 M). Protein refolding was
completed by incubation of the membranes for 3 h or overnight in
RNA binding buffer plus 0.1% Nonidet P-40. For RNA-protein complex
formation, the membranes were incubated for 1 h at room
temperature with 106 cpm/ml of 32P-labeled PVA
5'-untranslated region (+) transcript in the same buffer. The unbound
RNA was removed from the membranes by several 10-min washes with RNA
binding buffer containing 0.1% Nonidet P-40 and different
concentrations of NaCl (100, 300, or 500 mM). The
sufficient number of washes was determined by measuring the radioactivity of the discarded buffer. Finally, the membranes were
dried, and the remaining radioactively labeled RNA-protein complexes
were analyzed as described above.
RNA-Protein Binding Assays Using Metal Chelate Magnetic
Beads--
Prior to RNA-protein binding assays, in vitro
phosphorylation of His6-tagged recombinant protein was
carried out in the presence of 500 µM unlabeled ATP and
10 mM Mn2+ as described above. Phosphorylated
protein was checked for degradation by SDS-PAGE, and control reactions
were performed without the addition of plant protein extract. The
20-µl reaction mixtures containing 1 µg of phosphorylated or
nonphosphorylated protein were incubated with Ni2+-NTA
magnetic agarose beads (Qiagen) for 30 min at room temperature with
occasional swirling. The particle-protein complexes were separated from
unincorporated ATP and plant extract contaminants using a PickPen
magnetic particle transfer device (Bio-Nobile, Turku, Finland).
The beads were picked up from the solution, transferred to fresh tubes,
and washed with 150 µl of RNA binding buffer (50 mM
Tris-HCl, pH 8.0, 1 mM dithiothreitol, 50 mM
NaCl, 1 mg ml Calculations--
The total concentration of manganese ions
required for 95% saturation of ATP in the assay (0.5 µM)
was calculated using the Kd value taken from Ref.
23.
CP and VPg of PVA Are Phosphorylated in a Reconstituted System
Containing Protein Kinases from N. tabacum--
The four MRPs of PVA
(CIP, CP, HC-Pro, and VPg) and the MP of TMV were expressed in
Escherichia coli as fusion proteins with N-terminal
hexahistidine affinity tags and purified by immobilized metal affinity
chromatography. The Coomassie-stained gels presented in Fig.
1 show that the purified protein
preparations were free from any major contaminants. The purified PVA
MRPs were further assayed for phosphorylation in a reconstituted system
containing total plant protein extract and [
The VPg of PVA is covalently linked to the 5'-end of the viral genome,
whereas the CP of PVA is involved in the noncovalent interactions with
viral RNA in virions and putative movement intermediates. This apparent
difference suggests distinct functions for these two proteins in the
genome transport process, those of PVA CP more closely resembling the
functions attributed to TMV MP. Therefore, the current study was aimed
at comparison of the plant protein kinases involved in phosphorylation
of PVA CP and TMV MP and evaluation of a possible effect exerted by
phosphorylation on the RNA binding properties of PVA CP.
Effect of Staurosporine on PVA CP and TMV MP
Phosphorylation--
A powerful approach to dissect the functional
role of protein phosphorylation is to follow the change in the activity
of the target protein after specific inhibition of its phosphorylation. Therefore, our next goal was to determine an effective strategy for
interfering with the phosphorylation of viral MRPs. Staurosporine, which is a potent and broad spectrum inhibitor of protein kinases, was
introduced at 1 µM concentration into kinase assays
containing recombinant PVA CP and TMV MP, plant protein extracts, and
[ PVA CP Is Phosphorylated in Infected Plants--
Our next goal was
to verify that PVA CP is phosphorylated in PVA-infected tobacco leaves.
For this purpose, a series of experiments in which pieces of
PVA-infected leaves were incubated in [33P]orthophosphate
were performed. After overnight incubation, virus-infected cells were
lysed and immunoprecipitated with goat anti-PVA antibodies. The
resulting immunoprecipitates were analyzed on protein gel blots with
mouse anti-PVA IgG, followed by autoradiography. To determine the
phosphorylation status of in vivo synthesized PVA CP,
the radioactively labeled spots were superimposed on the specific spots
observed on protein gel blots. The 33P-labeled band with an
electrophoretic mobility similar to that of PVA CP was observed only in
immunoprecipitates of infected plants, demonstrating that it
corresponds to a phosphorylated, virus-encoded protein (Fig.
2B, lane 2). From these results, we concluded that PVA CP is phosphorylated in vivo. To further
support this conclusion, we tested the effect of staurosporine on PVA CP phosphorylation in infected plants. In agreement with the results obtained in vitro, phosphorylation of PVA CP in
vivo was also inhibited by 1 µM staurosporine (Fig.
2B, lane 3).
The Plant Protein Kinase Activity That Phosphorylates PVA CP Is
Ser/Thr-specific--
The substrate specificity of the plant protein
kinases phosphorylating the viral MRPs was previously determined only
for MPs of tobamoviruses. The phosphorylation sites within TMV MP
and tomato mosaic virus MP were mapped to serine or threonine residues (1, 10, 11). To identify the specificity of the enzyme(s) involved in
phosphorylation of PVA CP, we employed a two-step approach. As a first
step, we analyzed phosphorylated PVA CP (designated PVA pCP) by probing
protein gel blots with polyclonal phosphotyrosine-specific antibodies.
Phosphorylated TMV MP (TMV pMP), known to be modified at Ser/Thr, was
used in these experiments as a negative control. To verify that the
blotted proteins were indeed phosphorylated, we performed protein
kinase assays with radiolabeled ATP. Thus, the bands identified by
immunoblotting could be compared with the phosphorylation pattern. As
shown in Fig. 3A
(lanes 2 and 4), the
phosphotyrosine-specific antibodies did not recognize any of the bands
corresponding to phosphorylated PVA CP or TMV MP detected by
autoradiography. At the same time, the antibodies specifically
interacted with tyrosine-phosphorylated proteins from the epidermal
growth factor-stimulated A431 cell line lysate, which was used as a
positive control (Fig. 3A, lane 5).
Therefore, these data ruled out the possibility that the tyrosine
residues of PVA CP are phosphorylated. To obtain further evidence that PVA CP is phosphorylated on Ser/Thr, we employed a second strategy based on enzymatic dephosphorylation of recombinant proteins. PVA CP
was phosphorylated in vitro using radiolabeled ATP and then
treated with nonspecific SAP or PPTase-2A. Nonspecific alkaline phosphatases are known to strip the bound phosphate from any
phosphoester-containing compound including phosphoserine,
phosphothreonine, or phosphotyrosine. On the other hand, PPTase-2A
selectively hydrolyzes phosphoserine and phosphothreonine but does not
remove phosphate from phosphotyrosine. By comparing the extent of
radioactivity incorporated into PVA pCP before and after treatment with
these two phosphatases, it was possible to estimate the phosphorylation
state of target proteins. For this purpose, enzymatically
dephosphorylated PVA CP was analyzed by gel electrophoresis, and
incorporated label was visualized by autoradiography. One lane in each
dephosphorylation assay contained phosphoprotein incubated with
PPTase-2A in the presence of its potent inhibitor (1 µM
okadaic acid). A separate assay was performed with TMV pMP, which was
used as a Ser/Thr-phosphorylated positive control. Fig. 3B
shows that nearly all phosphorylation within PVA CP was reversed both
by SAP and by PPTase-2A, confirming that the protein is a substrate for
Ser/Thr-specific protein kinases. In the control lanes, the activity of
PPTase-2A was significantly inhibited by okadaic acid. Enzymatic assays
with TMV pMP produced similar results, with PPTase-2A having even
higher capacity to dephosphorylate this substrate than SAP. Thus, the
results obtained with phosphatase assays together with those obtained
by immunoblotting show that PVA CP is phosphorylated by plant protein
kinase activity with Ser/Thr-substrate specificity.
The Plant Protein Kinase Activity That Phosphorylates PVA CP and
TMV MP Exhibits a Preference for Mn2+ over Mg2+
and Is Not Ca2+-dependent--
It is well
established that protein kinases do not employ free ATP as a substrate
for protein phosphorylation, using instead the noncovalent complex
between ATP and divalent metal cation (phosphate-donating complex).
Therefore, the presence of divalent metal cation (DMC) is an
essential requirement for protein kinase activity. However, metal
cations can also play another important role in the catalysis through
direct or indirect (via water molecules) interaction with the enzyme
and/or coordination of the reaction intermediates. Because the
intracellular concentration of free DMCs is tightly regulated (24), the
enzymatic activity of certain protein kinases in the cell may be
controlled through dynamic changes in the local concentration of metal
ions (25). One of the purposes of the present study was to examine the
divalent cation requirement for the plant protein kinase activity
phosphorylating viral MRPs. It was found that phosphorylation of PVA CP
by the cellular protein kinase(s) from total protein extract of tobacco was stimulated by Mn2+. The dependence of kinase activity
on the concentration of Mn2+ is shown in Fig.
4A. Maximal activity was
achieved at about 1 mM of MnCl2, and a minor
inhibition of protein phosphorylation was observed at concentrations of
MnCl2 higher than 1 mM. Such a decrease in
activity may be due to the nonspecific interaction between metal ion
and protein that is often observed at concentrations higher than 5 mM (26), reducing the amount of free DMC in the assay. When
free Mn2+ was removed from the phosphorylation reaction
mixture in a complex with 10 mM EDTA, the kinase activity
dropped down to the control level (Fig. 4A).
To determine the cation specificity of the plant protein kinase
activity, we compared levels of PVA CP and TMV MP phosphorylation at
the same 10 mM concentrations of Mn2+,
Mg2+, and Ca2+. The results presented in Fig.
4B demonstrate that the plant protein kinase activity
exhibited a clear preference for Mn2+ over Mg2+
with each of the substrate proteins. However, in contrast to Mn2+ and Mg2+, the addition of 10 mM Ca2+ into the assays did not stimulate
substrate protein phosphorylation (Fig. 4B). This is in
agreement with the recent report (3) that protein kinase activity
phosphorylating TMV MP requires the presence of Mg2+ but
not Ca2+ cations.
Micromolar Concentrations of Zn2+ and Cd2+
Inhibit the Mg2+-dependent Phosphorylation of
PVA CP and TMV MP--
It is well recognized that, compared with
Mn2+ and Mg2+, Zn2+ and
Cd2+ have remarkably different effects on the activity of
several protein kinases including Csk, a soluble protein-tyrosine
kinase. While Mn2+ and Mg2+ function as
essential activators of these enzymes, Zn2+ and
Cd2+ strongly inhibit them. In the case of Csk,
Zn2+, and Cd2+, concentrations as low as 10 µM were found to inhibit enzymatic activity by more than
95% in solutions that already contain high concentrations of
Mg2+ (23). In the current study, we monitored
phosphorylation of two substrate proteins (PVA CP and TMV MP) at
increasing concentrations of zinc or cadmium (0 µM, 10 µM, 100 µM, and 1 mM) in the
presence of a large excess (10 mM) of Mg2+. It
is important to note that since micromolar concentrations of
Zn2+ and Cd2+ were used in these experiments,
precautions were made to avoid nonspecific binding of these cations to
ATP, substrate proteins, and buffer constituents. Zn2+ and
Cd2+ were always the last components added to the assay
together with the plant protein extract (kinase source), while ATP was
already saturated with Mg2+, and buffers were made with
HEPES rather than Tris. Fig. 5 shows that
both Zn2+ and Cd2+ acted as inhibitors of
protein kinase activity. The addition of 10 µM
Zn2+ or Cd2+ in the presence of 10 mM Mg2+ already markedly reduced
phosphorylation levels of both substrate proteins: PVA CP and TMV MP.
The 1000-fold difference in concentrations of zinc and cadmium (10 µM) and magnesium (10 mM) ensured that the
actual concentrations of free Zn2+ and Cd2+
were not affected by complexing with ATP.
Thus, we concluded that PVA CP and TMV MP are both phosphorylated by
the same type of plant protein kinase activity having a distinct mode
of kinetic response to metal cations. This enzymatic activity is not
Ca2+-dependent but shows a clear preference for
Mn2+ over Mg2+ and may be inhibited by very low
concentrations of Zn2+ and Cd2+. To further
support this conclusion, competition assays were performed in which PVA
CP and TMV MP were together incubated in a reconstituted system
containing plant protein kinases and [ PVA CP and TMV MP Are Competing Substrates for the Same
Mn2+-activated Plant Protein Kinase Activity--
To
determine whether PVA CP and TMV MP are phosphorylated by the same
plant enzyme, constant amounts of each protein were assayed for
phosphorylation in the presence of increasing amounts of the other
substrate protein. Two types of experiments were carried out. In one
set of experiments, kinase assays contained a constant amount of TMV MP
and increasing concentrations of PVA CP. Alternatively, phosphorylation
reactions were performed with a constant amount of PVA CP and
increasing concentrations of TMV MP. Following incubations with plant
protein extracts in the presence of 10 mM Mn2+,
the radiolabeled phosphoproteins were separated by gel electrophoresis, transferred to membranes, and autoradiographed. Fig.
6A shows that at higher
competitor concentrations, less label was incorporated into the
substrate proteins. These results strongly suggest that PVA CP and TMV
MP are able to compete with each other as substrates for the same
Mn2+-activated protein kinase. To eliminate the possibility
that PVA CP and TMV MP are phosphorylated by a plant protein kinase
with low specificity, we compared the ability of another PVA-encoded protein, VPg, to compete for phosphorylation with PVA CP and TMV MP.
The putative movement complex of PVA is composed of CP and viral RNA,
which, in turn, has VPg covalently attached to its 5'-end. Therefore,
VPg is likely to colocalize with CP in the same subcellular
compartments during virus assembly and movement. Should there be a
protein kinase having low specificity and using several proteins as
substrates, it would phosphorylate not only CP but also VPg. On the
contrary, the results presented in Fig. 6B show that PVA VPg
could not compete as a kinase substrate with either PVA CP or TMV MP.
This finding allowed us to conclude that the phosphorylation of PVA CP
and TMV MP by the Mn2+-activated plant protein kinase
activity is substrate-specific and that PVA VPg is a substrate for
another kinase.
Comparison of the Phosphorylation Patterns of the PVA CP Purified
from Bacteria with That of the Virion-extracted
Protein--
One of the key questions raised in studies employing
bacterially expressed proteins is whether the polypeptide obtained from bacteria is folded similarly to the native protein synthesized in an
eukaryotic system. This point is of extreme importance in the current
study, since the location of specific phosphorylation sites strongly
depends on the protein conformation. To minimize its effects on protein
folding, we introduced the shortest possible histidine tag into the
recombinant proteins. In addition, purified proteins were tested for
their ability to bind RNA to verify that they preserve active
conformations. However, additional evidence was required to prove that
the bacterially expressed protein is correctly folded and, therefore,
has a similar phosphorylation pattern with the protein synthesized
in vivo. For this purpose, PVA CP was extracted by LiCl from
virus particles and phosphorylated in a reconstituted system in
parallel with the bacterially expressed protein. Tryptic peptide
analysis of the resulting phosphoproteins revealed that the
phosphorylation patterns of the bacterially expressed and the
virion-extracted PVA CP were nearly identical. As shown in Fig.
7, five spots with similar mobilities
were detected on both peptide maps, suggesting that PVA CP is
phosphorylated at multiple sites. However, one extra spot (marked VI)
was reproducibly observed on the peptide map of the in vivo
synthesized coat protein. This spot had a very low chromatographic
mobility, suggesting that it may correspond to a hydrophilic nucleic
acid derivative. Nevertheless, we cannot completely discount it as a
nucleotide contaminant and rule out the possibility that it represents
a phosphopeptide. Despite this one possible contradiction, we conclude from the comparison of the peptide maps that the bacterially expressed and the in vivo synthesized proteins share the same
conformation and are similarly phosphorylated by the plant protein
kinase activity.
PVA CP Is Not Phosphorylated When Packaged into Virions--
To
determine whether virion formation has any effect on PVA CP
phosphorylation, in vitro kinase assays with purified virus particles were performed. As shown in Fig.
8 (lane 2),
autoradiography revealed no bands corresponding to
33P-labeled phosphorylated PVA CP. Two possible
explanations for this finding are that PVA CP is already extensively
phosphorylated in virions or its phosphorylation is blocked upon virus
packaging. To investigate the first possibility, we extracted the coat
protein from the same virion preparation and again examined its
phosphorylation. For this purpose, the virus was disassembled in
guanidine HCl or LiCl, and the released CP was tested in kinase assays.
Fig. 8A (lanes 4 and 6)
shows that both guanidine HCl-extracted and LiCl-extracted CP were
effectively phosphorylated in vitro by the plant protein
kinase activity. Thus, we ruled out the possibility that the absence of
detectable phosphorylation of intact virions was simply the result of
preliminary phosphorylation of the coat protein in infected plants.
Taken together, the results presented above strongly suggest that PVA
CP is not phosphorylated when packaged into virions. This fact may be
explained by the existence of conformational restrictions preventing
the exposure of the PVA CP phosphorylation sites upon virion assembly.
One possibility is that the CP phosphorylation sites are not exposed on
the surface of the virus particle because they are located within the
core domain involved in interaction with the viral RNA. Multiple
alignments of the amino acid sequences of the potyviral coat proteins
revealed at least 10 consensus Ser/Thr residues within the highly
conserved core region (27, 28). Therefore, we suggested that
phosphorylation of some Ser/Thr residues may potentially affect the RNA
binding properties of the coat protein. To address this possibility, we next studied the effect of phosphorylation on the RNA binding properties of PVA CP using two different experimental approaches.
Phosphorylation Down-regulates Binding of PVA CP to RNA--
To
begin our analyses, we implemented a multistep technique based on
blotting of phosphorylated PVA CP and probing it with radioactively
labeled RNA transcript (RNA-protein blotting). The procedure comprised
five stages: 1) target protein was phosphorylated in the presence of
Mn2+ in a reconstituted system containing plant protein
kinases from total cellular extract; 2) the phosphoprotein was
separated from unincorporated ATP and protein extract contaminants by
SDS-PAGE and transferred to membranes; 3) SDS was removed from the
blotted protein by guanidine HCl extraction; 4) immobilized protein was renatured and incubated with radiolabeled RNA transcript; 5) unbound RNA was washed from the membranes with a NaCl solution, and the remaining RNA-protein complex was visualized by autoradiography.
An important aspect of the above assay system is the complete removal
of unincorporated ATP from the phosphorylation reaction mixture by gel
electrophoresis prior to analysis of the target protein. Furthermore,
successful detection of the effect of phosphorylation on RNA binding
largely depends on the final amount of the phosphoprotein in the assay.
As long as purified preparations of the plant enzymes involved in MRP
phosphorylation are not available, the only sources of protein
kinases for the assays are cellular extracts that contain natural
protein kinase inhibitors and protein phosphatases. Therefore, the
yield of phosphorylated protein in such assays may be insufficient and
should be artificially increased. This can be achieved by inhibition of
the protein phosphatase activity using polyspecific inhibitors such as
fluoride ion or by direct stimulation of the plant protein kinase
activity. We determined that a stimulation of the plant protein kinase
activity by Mn2+ is the most straightforward and efficient
strategy to obtain a large pool of phosphorylated MP molecules.
The results obtained with the technique described above demonstrated
that the RNA binding activity of PVA CP was strongly affected by
phosphorylation. Fig. 8B shows that the affinity of phosphorylated PVA CP for RNA was remarkably lower compared with that
of nonphosphorylated protein. Densitometry revealed that the RNA
binding affinity of phosphorylated protein dropped about 100-fold
compared with that of nonphosphorylated protein. To ensure that no
other protein modification except phosphorylation was responsible for
the observed effect, we performed a control assay in which
staurosporine was introduced into the phosphorylation reaction
mixtures. Because staurosporine was previously shown to inhibit
phosphorylation of PVA CP, its presence in the kinase assay was thought
to preserve protein functions affected by phosphorylation. Indeed,
the RNA binding activity of PVA CP remained at the control level after
the protein was subjected to mock treatment with the plant protein
extract in the presence of 1 µM staurosporine (Fig. 8C).
To further support the conclusion that phosphorylation down-regulates
the RNA binding function of PVA CP, we implemented another experimental
approach. It has been demonstrated that His6TMV MP can
effectively bind RNA while immobilized on the column with Ni2+-NTA adsorbent (19). In this study, we developed a
convenient modification of this method based on the magnetic bead
technology. This approach combined the advantages of the noncovalent
protein immobilization with the ease and speed of magnetocapture
assays. First, the target His6PVA CP was extensively
phosphorylated in a reconstituted system containing plant protein
extract, unlabeled ATP, and Mn2+ as an essential protein
kinase activator. The phosphorylated protein was next immobilized on
the Ni2+-NTA magnetic agarose beads in parallel with the
negative control, the nonphosphorylated protein. The beads were
magnetically separated from unincorporated ATP and contaminating plant
proteins, washed with RNA binding buffer, and incubated with
radiolabeled RNA transcript. Further washings removed free RNA from the
beads, and the remaining radioactivity was measured by liquid
scintillation counting. Control experiments with no immobilized protein
indicated that RNA itself does not bind to the beads. It is important
to note that the RNA binding buffer contained BSA to prevent
nonspecific interactions, and PVA CP was checked for degradation by gel
electrophoresis (Fig. 8D, inset). In agreement
with the results obtained by RNA-protein blotting, analysis using
magnetic beads also revealed that the RNA binding of PVA CP was
strongly down-regulated by phosphorylation (Fig. 8D).
In recent years, the role of MP phosphorylation in virus movement
has began to be revealed. There is a growing body of evidence that MP
phosphorylation represents a cellular function that controls spread of
the virus (1-3). In the current study, we demonstrate that
phosphorylation by the plant protein kinase activity with a distinct
mode of activation and inhibition by divalent metal cations strongly
down-regulates interactions of PVA CP with RNA. Furthermore, we provide
evidence that the same protein kinase activity phosphorylates the well
characterized TMV MP. Our results indicate that this activity can be
highly stimulated by the manganese cations. The possible mechanism
behind such stimulation, at first sight, seems obvious; increasing
concentrations of Mn2+ can activate the enzyme catalytic
activity through a noncovalent interaction with ATP and the formation
of the ATP-DMC complex, which is the true donor of phosphate for
protein kinases. However, the concentrations of Mn2+ used
in our assays were well above the limit necessary to completely saturate 0.5 µM ATP in the reaction mixture. We
calculated that 95% saturation of 0.5 µM ATP is achieved
at 0.19 mM Mn2+. If metal cations can affect
catalytic activity only through an interaction with ATP, there will be
no stimulation of phosphorylation after all ATP molecules become
saturated with Mn2+. On the contrary, our results show that
the plant kinase activity phosphorylating PVA CP rises further when
Mn2+ concentrations increase beyond those needed to
saturate ATP. This suggests that in addition to interaction with ATP,
divalent metal cations play another important role in the activation of this plant enzyme. Previously, the problem of kinase activation by DMCs
has been extensively studied for several animal tyrosine kinases
including Csk, Src, and the fibroblast growth factor receptor kinase
(23, 25). The catalytic activity of these enzymes has been also shown
to depend on the concentration of DMC in the assay at levels higher
than those required to completely saturate ATP. This led to the
conclusion that protein-tyrosine kinases possess a second metal binding
site that is essential for enzyme activation (25). It has been
suggested that an additional metal cation can activate the kinase by
binding directly to the enzyme or via a metal-substrate complex. In the
course of the current study, we observed that the plant protein kinase
that phosphorylates PVA CP and TMV MP is activated in a fashion similar
to Csk and Src at concentrations of Mn2+ higher than those
required for ATP saturation. This suggests that the plant enzyme also
requires additional divalent metal cations as essential activators.
Similarly to tyrosine kinase Csk, the plant Ser/Thr kinase activity
involved in MRP phosphorylation was also inhibited by micromolar
concentrations of Zn2+ and Cd2+ in the presence
of a 1000-fold excess of Mg2+. Taken together, these
results suggest that the plant enzyme involved in PVA CP and TMV MP
phosphorylation as well as certain animal protein kinases including Csk
may share the common functional domains responsible for the regulation
of catalytic activity through interactions with metal ions.
Interestingly, inhibition of protein phosphorylation by cadmium
suggests a possible mechanism behind the recently reported
cadmium-induced resistance of tobacco plants to turnip vein clearing
virus (genus Tobamovirus) (29, 30).
The noncovalent binding to RNA represents one of the essential
functions attributed to the movement-related proteins of plant viruses.
An elegant hypothesis that the translocation of viral RNA through
plasmodesmata occurs in the form of vRNP complexes was put forward
after it was shown that TMV MP strongly interacts with
single-stranded nucleic acids in vitro (31). Recently, major
support for the assumption that vRNP is indeed a movement intermediate
was gained when it was shown that viral RNA colocalizes with TMV MP in
infected tobacco protoplasts (32). It is assumed that MP has the
capacity to unfold viral RNA, creating vRNP complexes that are
structurally compatible with modified plasmodesmata (33, 34). The
finding that TMV MP serves as an efficient repressor of TMV RNA
translation in vitro (35) suggested an interesting possibility that a strong interaction between MP and viral RNA switches
the RNA function from replication to movement. In other words, viral
RNA-MP complexes may represent movement intermediates excluded from
replication and destined solely for cell-to-cell translocation. This
hypothesis, in turn, raised another question: what is the mechanism
converting the translation-incompetent movement intermediates back into
the infectious form when they reach the neighboring cell? The possible
answer to this question emerged when phosphorylation of TMV MP was
shown to abolish its ability to repress RNA translation in
vitro (2). It has been suggested that MP phosphorylation
represents a molecular strategy to restore translation of viral RNA in
the newly infected cell.
Although the cell-to-cell movement of tobamoviruses is assumed to occur
in a form of vRNP complexes, the nature of the transport complex of
potyviruses is still largely unknown. One possibility is that
potyviruses move from cell to cell as ribonucleoprotein complexes,
analogous to that proposed for TMV but containing the coat protein
(36). If the latter is true, a similar phosphorylation-mediated mechanism may exist to control the stability of potyvirus movement intermediates. Our finding that phosphorylation of PVA CP strongly inhibits its RNA binding function suggests to us a hypothetical model
describing how the function of the PVA movement intermediates is
regulated by phosphorylation. According to this model, at middle and
late stages of infection, a certain pool of viral RNA becomes excluded
from replication through a strong cooperative interaction with CP and
then is targeted to structurally modified plasmodesmata. The unfolded
vRNP movement intermediates are then actively transported through
plasmodesmata into the neighboring cells. During or after translocation
through plasmodesmata, cellular protein kinases phosphorylate CP
molecules, decreasing their affinity toward viral RNA. Consequently,
the translation-incompetent vRNP movement intermediates dissociate,
allowing the viral RNA to start replication in the newly infected cell.
Alternatively, potyviruses may require virion assembly for cell-to-cell
translocation. In this case, phosphorylation may control movement of
potyviruses by regulating the amount of CP available for interaction
with viral RNA. Our results demonstrate that the protein extracted from
virions but not the virions themselves could be phosphorylated. This
finding suggests that phosphorylation may sequester a certain amount of
nonvirion CP from interactions with viral RNA, thus regulating the
process of virus packaging.
What is the molecular mechanism behind the phosphorylation-mediated
regulation of the RNA binding properties of PVA CP? The most
straightforward explanation requires that multiple phosphorylations add
extra negative charges to the RNA binding domains of the protein and
inhibit its electrostatic interactions with RNA. However, regulation of
PVA CP function by phosphorylation may be a more complex process. It
may not be ruled out that phosphorylation causes a conformational
change within the target protein. The fact that phosphorylation of PVA
CP produced a very strong effect suggests that its function is probably
affected by phosphorylation on a structural level. Ultimately,
three-dimensional crystal structures of movement-related proteins will
be required to determine the effect of phosphorylation on their domain conformations.
Finally, still another role played by phosphorylation in viral movement
may be proposed. Specific interactions of MPs with cellular proteins
having the characteristics of plasmodesmal receptors have been recently
reported (37-39). It needs to be established whether specific
phosphorylation events control functions of these proteins that
potentially regulate the cell-to-cell molecular pathway. There is a
possible correlation between the developmental regulation of cell
wall-associated plant protein kinase activity (11) and the transition
from simple to branched forms of plasmodesmata (40). Assuming that
protein phosphorylation is involved in specific macromolecular
trafficking through plasmodesmata, changing patterns of protein kinase
expression in the leaves undergoing the sink-source transition may
reflect a part of a complex developmental strategy used by plants to
regulate plasmodesmal permeability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside-inducible
expression of N-terminal His6 fusion proteins.
1) of [33P]orthophosphate
(Amersham Pharmacia Biotech;
3000 Ci/mmol) in the presence or absence
of 1 µM staurosporine (Sigma). Vacuum was applied until
the leaf discs darkened and the mixture was further incubated overnight
at 22 °C. Following removal of the incubation solution, the leaf
discs were thoroughly rinsed, dried on filter paper, and cut into
smaller pieces. The resulting leaf fragments were homogenized in NET
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Nonidet P-40 (Sigma),
0.02% NaN3, 100 units ml
1 of
Trasylol (Bayer)) containing 1% SDS and immediately boiled for 15 min.
The lysates were cleared by centrifugation and diluted (1:10) with NET
buffer for immunoprecipitation. Presoaked protein A-Sepharose (Amersham
Pharmacia Biotech) was added at 1% (w/v) to the diluted lysates and
incubated for 1.5 h at 4 °C to remove Sepharose-binding
proteins. Following centrifugation at 3200 × g for 10 min at 4 °C, sheep anti-PVA antibody (Roche Molecular Biochemicals)
was added to the supernatants and incubated overnight at 4 °C with
agitation. The protein-antibody complexes were then allowed to interact
with protein A-Sepharose for 2 h at 4 °C and centrifuged at
3200 × g for 10 min at 4 °C. After removal of
supernatants, the pellets were washed five times with NET buffer, and
the Sepharose-bound proteins were resolved by SDS-PAGE and
electrotransferred to Immobilon-P membranes. The membranes were probed
with mouse anti-PVA IgG (Bioreba) as described above and autoradiographed.
-33P]ATP into the
purified substrate proteins. Redivue [
-33P]ATP (
2500
Ci/mmol) was obtained from Amersham Pharmacia Biotech. Assays were
performed at room temperature for 30 min with occasional swirling in a
final volume of 15 µl containing 0.5 µM
[
-33P]ATP (~10 µCi), 1 µg of substrate protein,
25 mM HEPES, pH 7.4, in the presence or absence of divalent
cations (Mg2+, Mn2+, Ca2+,
Zn2+, and Cd2+ at the indicated
concentrations). Unless stated, freshly prepared total plant protein
extract (
3 µg) was used as a kinase source. For Western analysis
with anti-phosphotyrosine antibody, the kinase assays were performed
with 20 µCi of [
-33P]ATP together with 50 µM unlabeled ATP. To study the effect of staurosporine on
protein phosphorylation, the compound was added into the kinase assays
at a final concentration of 1 µM. Reactions were
terminated by adding 5 µl of 5× SDS-PAGE sample buffer, followed immediately by boiling for 5 min. The phosphorylated proteins were
analyzed by SDS-PAGE as described above.
1 BSA, 10% glycerol (v/v)). The
procedure was repeated again, and the pellet was resuspended in 50 µl
of RNA binding buffer. The beads were then incubated with 5 × 105 cpm of 32P-labeled PVA 5'-untranslated
region (+) transcript for 20 min on ice with occasional gentle shaking.
Two more washes with RNA binding buffer followed each time by particle
transfer to fresh tubes were performed. This procedure removed the
unbound RNA from the beads. Finally, the beads were resuspended in 50 µl of RNA binding buffer and the residual radiolabel was checked
using a liquid scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-33P]ATP.
The phosphorylation reaction mixtures were subjected to SDS-PAGE
followed by staining with Coomassie Brilliant Blue and autoradiography.
The 30-kDa MP of TMV was used as a positive control in all assays,
since its phosphorylation by plant protein kinases is well
characterized. Two negative controls were performed in each assay to
verify that recombinant proteins do not themselves bind radiolabeled
ATP and that bands on the autoradiogram do not correspond to cellular
phosphoproteins. In these control experiments, either purified
recombinant proteins or plant protein extracts were alone incubated in
the presence of [
-33P]ATP. As shown in Fig. 1, two
MRPs of PVA, CP and VPg, were found to be phosphorylated by plant
protein kinases in the reconstituted system, but the phosphorylation of
two other proteins, CIP and HC-Pro, was not detected. Autoradiography
did not reveal any labeled protein in the negative controls, confirming
that bands identified in other lanes correspond to phosphorylated
recombinant proteins. As expected, TMV MP was phosphorylated in all
control reactions, showing that plant extracts used in the assays
contained active protein kinases (Fig. 1, lanes
1).
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Fig. 1.
In vitro phosphorylation of the
MRPs of PVA. Bacterially expressed His-tagged CIP (A),
CP (B), HC-Pro (C), and VPg (D) were
assayed for phosphorylation in the presence of
[ -33P]ATP as described under "Experimental
Procedures." Proteins were separated by 12% SDS-PAGE and stained
with Coomassie Blue (left column), and
phosphorylation was detected by autoradiography (right
column). Two of the four PVA MRPs, CP (B,
lane 2) and VPg (D, lane
2), were found to be phosphorylated by protein kinase
activity from tobacco leaves. The movement protein of tobacco mosaic
virus (TMV MP) was used in each panel as a positive control for
phosphorylation (lane 1). To ensure that detected
bands did not correspond to cellular phosphoproteins, plant extracts
were incubated alone in the presence of
[
-33P]ATP (A, lane 3;
B, C, and D, lanes
4). Alternatively, to rule out NTP binding and
autophosphorylation, substrate proteins were incubated with
[
-33P]ATP in the absence of plant extract
(A, lane 4; B,
C, and D, lanes 3). The
positions of molecular mass markers (lane M) are
indicated in kilodaltons on the left of each
panel.
-33P]ATP. Fig.
2A shows that staurosporine
had an inhibitory effect on the phosphorylation of both studied
proteins. This finding allowed us to use staurosporine in the
subsequent experiments as an inhibitor of PVA CP phosphorylation.
View larger version (24K):
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Fig. 2.
A, staurosporine inhibits
phosphorylation of PVA CP and TMV MP. Equal amounts of bacterially
expressed His-tagged proteins were phosphorylated in a reconstituted
system containing [ -33P]ATP in the absence (
) or
presence (+) of 1 µM staurosporine. The phosphoproteins
were separated by 12% SDS-PAGE and transferred to membranes, and their
positions were identified by staining with Ponceau S. The radioactive
bands were visualized by autoradiography. B, PVA CP is
phosphorylated in infected tobacco plants. Leaves of mock-infected
(B, lane 1) or PVA-infected
(B, lanes 2 and 3) plants
were metabolically labeled with [33P]orthophosphate in
the absence (
) or presence (+) of 1 µM staurosporine as
described under "Experimental Procedures." SDS-treated cell lysates
were immunoprecipitated with goat anti-PVA antibodies. The resulting
immunoprecipitates were analyzed by probing protein gel blots with
mouse anti-PVA IgG (B, left panel) and
subjected to autoradiography (B, right
panel). The positions of molecular mass markers are
indicated in kilodaltons on the left of the lower
panel.
View larger version (38K):
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Fig. 3.
Phosphorylation of PVA CP by the plant
protein kinase activity is Ser/Thr-specific. A, PVA CP
and TMV MP were incubated in a reconstituted system containing a
mixture of 33P-labeled and unlabeled ATP. The
nonphosphorylated ( ) and phosphorylated (+) proteins were separated
by 12% SDS-PAGE and analyzed for phosphotyrosine on protein gel blots.
While tyrosine-phosphorylated proteins from epidermal growth
factor-stimulated A431 cell line lysate (lane 5)
were detected on the blots, none of the viral movement-related proteins
was recognized by the anti-phosphotyrosine antibodies (right
panel). Phosphorylation of all proteins was confirmed by
autoradiography (center panel). The positions of
molecular mass markers (lane M) are indicated in
kilodaltons. B, in an alternative approach, radioactively
labeled phosphoproteins (PVA pCP and TMV pMP) were enzymatically
dephosphorylated by nonspecific SAP and Ser/Thr-specific PPTase-2A. In
the control lanes, dephosphorylation of the substrate proteins by
PPTase-2A was inhibited by 1 µM okadaic acid
(OA). All proteins shown are histidine fusions.
View larger version (30K):
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Fig. 4.
A, phosphorylation of PVA CP by plant
protein kinase activity is stimulated by Mn2+.
Increasing concentrations of manganese were added into assays
containing bacterially expressed protein, total protein kinase activity
from tobacco leaves, and [ -33P]ATP. Phosphoproteins
were separated by 12% SDS-PAGE and transferred to membranes, and their
positions were identified by staining with Ponceau S or Amido Black.
Radioactivity associated with phosphoproteins was quantitated with a
phosphor imager and plotted against Mn2+
concentration. In a control experiment, manganese was removed from
phosphorylation reaction by the addition of 10 mM EDTA.
B, effect of Mn2+, Mg2+, or
Ca2+ on phosphorylation of PVA CP and TMV MP. Proteins were
assayed for phosphorylation in a reconstituted system containing plant
enzymes, [
-33P]ATP, and 10 mM
Mn2+, Mg2+, or Ca2+. Proteins were
subjected to SDS-PAGE and transferred to membranes. Autoradiograms of
phosphorylated proteins are shown together with stained membranes. All
proteins shown are histidine fusions.
View larger version (41K):
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Fig. 5.
Micromolar concentrations of Zn2+
(A) and Cd2+ (B) inhibit
phosphorylation of PVA CP and TMV MP in the presence of 10 mM Mg2+. Effect of Zn2+ and
Cd2+ on PVA CP and TMV MP phosphorylation was studied in
assays containing indicated concentrations of metal ions, bacterially
expressed proteins, total protein kinase activity from tobacco leaves,
and [ -33P]ATP. Phosphoproteins were separated by 12%
SDS-PAGE and transferred to membranes, and their positions were
identified by staining with Ponceau S or Amido Black. Radioactivity
associated with phosphoproteins was quantitated with a phosphor
imager and plotted against Zn2+ or Cd2+
concentration. All proteins shown are histidine fusions.
-33P]ATP.
View larger version (32K):
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Fig. 6.
A, PVA CP and TMV MP
compete as substrates for the same Mn2+-activated plant
protein kinase. Constant amounts of TMV MP (120 ng,
left panel) or PVA CP (50 ng, right
panel) were incubated in a reconstituted system containing
total plant protein kinase activity, [ -33P]ATP,
manganese cations, and increasing amounts of their respective
competitors. The following competitor levels were used: 0, 5, 50, 250, and 700 ng of PVA CP (left) or 0, 12, 120, 600, and 1680 ng
of TMV MP (right). Proteins were separated by 12% SDS-PAGE,
transferred to membranes, and stained with Amido Black, and their
phosphorylation was detected by autoradiography. The incorporation of
radioactive label into the phosphorylated proteins was quantitated with
a phosphor imager and plotted against the amounts of competitor
protein. B, PVA VPg is unable to compete as a kinase
substrate with either PVA CP or TMV MP. The competition experiments
were performed as described for A except that PVA VPg was
assayed for phosphorylation together with PVA CP or TMV MP. All
proteins shown are histidine fusions. The lower
panels are overstained to visualize ng of protein.
View larger version (26K):
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Fig. 7.
Comparison of tryptic peptide maps of
bacterially expressed and virion-extracted PVA CP. The coat
protein was extracted from virions and phosphorylated in a
reconstituted system in the presence of 10 mM
Mn2+ in parallel with the bacterially expressed protein.
The phosphorylated proteins were gel-purified, transferred to
membranes, and subjected to trypsin digestion. The digests were
lyophilized and separated by thin layer electrophoresis in the
first dimension and by chromatography in the second. The positions of
phosphopeptides were identified by autoradiography (upper
panel) or using a phosphor imager (lower
panel).
View larger version (39K):
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Fig. 8.
A, PVA CP is not phosphorylated when
packaged into virions. PVA CP in intact virions (lane
2) was assayed for phosphorylation in parallel with equal
amounts of the coat protein extracted from the same virion preparation
using guanidine HCl (Gu HCl; lane 4)
or LiCl (lane 6) methods. Bacterially expressed
PVA CP was used as a positive control for phosphorylation
(lane 8). Control reactions without the addition
of plant protein extracts (lanes 1, 3,
5, and 7) were performed. The lower
panel represents a Coomassie-stained gel of CP amounts used
in the experiment. B, binding of PVA CP to RNA is regulated
by phosphorylation. PVA CP was phosphorylated in vitro in
the presence of unlabeled ATP and 10 mM MnCl2.
The phosphoprotein (lane 2) was separated from
unincorporated ATP by SDS-PAGE, transferred to membrane, and probed
with 32P-labeled RNA transcript. Control
lanes 1 and 3 contained the
nonphosphorylated PVA CP and BSA. After protein transfer, the membranes
were either stained with Amido Black or incubated with radioactively
labeled RNA. The positions of molecular mass markers (lane
M) are indicated in kilodaltons. C, the addition
of 1 µM staurosporine into the protein kinase assay
preserves the RNA binding activity of PVA CP. The protein incubated
with plant extract in the presence of staurosporine (+/+) possesses the
same high affinity to RNA as the unphosphorylated protein ( /
). In
the control lane, protein was phosphorylated in the absence of
staurosporine (+/
), and its binding to RNA was inhibited.
D, analysis of the effect of protein phosphorylation on the
RNA binding properties of PVA CP using magnetic beads (see
"Experimental Procedures"). Graphic representation of the results
obtained from three independent experiments. Each bar
represents mean ± S.E. values. Phosphorylated protein (+) was
checked for degradation by SDS-PAGE and compared with nonphosphorylated
protein (
) (amounts of protein are shown in the inset).
All proteins shown are histidine fusions except in A,
lanes 1-6.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Kirk Overmyer, Dr. Alan Schulman, Dr. Deyin Guo, and Jimmy Lucchesi for critical reading of the manuscript. We are grateful to Helena Vihinen and Dr. Igor Fabrichny for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by Academy of Finland Grant 40934, EC INCO Copernicus Program Grant IC 15-CT97-0900, and Biocentrum Helsinki.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Fellow of Biocentrum Helsinki.
§ To whom correspondence should be addressed. Tel.: 358 9 19159573; Fax: 358 9 19159571; E-mail: Kristiina.Makinen@helsinki.fi.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009551200
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ABBREVIATIONS |
---|
The abbreviations used are: MP, movement protein; TMV, tobacco mosaic virus; MRP, movement-related protein; CP, coat protein; HC-Pro, helper component protease; CIP, cylindrical inclusion protein; PVA, potato virus A; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; SAP, shrimp alkaline phosphatase; PPTase-2A, protein phosphatase-2A; DMC, divalent metal cation.
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REFERENCES |
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---|
1. |
Kawakami, S.,
Padgett, H. S.,
Hosokawa, D.,
Okada, Y.,
Beachy, R. N.,
and Watanabe, Y.
(1999)
J. Virol.
73,
6831-6840 |
2. | Karpova, O. V., Rodionova, N. P., Ivanov, K. I., Kozlovsky, S. V., Dorokhov, Yu. L., and Atabekov, J. G. (1999) Virology 261, 20-24[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Waigmann, E.,
Chen, M.-H.,
Bachmaier, R.,
Ghoshroy, S.,
and Citovsky, V.
(2000)
EMBO J.
19,
4875-4884 |
4. |
Carrington, J. C.,
Kasschau, K. D.,
Mahajan, S. K.,
and Schaad, M. C.
(1996)
Plant Cell
8,
1669-1681 |
5. |
Lazarowitz, S. G.,
and Beachy, R. N.
(1999)
Plant Cell
11,
535-548 |
6. | Santa Cruz, S. (1999) Trends Microbiol. 7, 237-241[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lee, J.-Y., Yoo, B.-C., and Lucas, W. J. (2000) Planta 210, 177-187[Medline] [Order article via Infotrieve] |
8. | Atkins, D., Roberts, K., Hull, R., Prehaud, C., and Bishop, D. H. L. (1991) J. Gen. Virol. 72, 2831-2835[Abstract] |
9. | Watanabe, Y., Ogawa, T., and Okada, Y. (1992) FEBS Lett. 313, 181-184[CrossRef][Medline] [Order article via Infotrieve] |
10. | Haley, A., Hunter, T., Kilberstis, P., and Zimmern, D. (1995) Plant J. 8, 715-724[CrossRef][Medline] [Order article via Infotrieve] |
11. | Citovsky, V., McLean, B. G., Zupan, J., and Zambryski, P. (1993) Genes Dev. 7, 904-910[Abstract] |
12. | Sokolova, M., Prüfer, D., Tacke, E., and Rohde, W. (1997) FEBS Lett. 400, 201-205[CrossRef][Medline] [Order article via Infotrieve] |
13. | Seron, K., Bernasconi, L., Allet, B., and Haenni, A.-L. (1996) Virology 219, 274-278[CrossRef][Medline] [Order article via Infotrieve] |
14. | Hughes, R. K., Perbal, M.-C., Maule, A. J., and Hull, R. (1995) Mol. Plant-Microbe Interact. 8, 658-665[Medline] [Order article via Infotrieve] |
15. | Revers, F., Le Gall, O., Candresse, T., and Maule, A. J. (1999) Mol. Plant-Microbe Interact. 12, 367-376 |
16. | Hammond, J., and Lawson, R. H. (1988) J. Virol. Methods 20, 203-217[Medline] [Order article via Infotrieve] |
17. | McDonald, J. G., and Bancroft, J. B. (1977) J. Gen. Virol. 35, 251-263 |
18. | Merits, A., Guo, D., and Saarma, M. (1998) J. Gen. Virol. 79, 3123-3127[Abstract] |
19. | Ivanov, K. I., Ivanov, P. A., Timofeeva, E. K., Dorokhov, Yu. L., and Atabekov, J. G. (1994) FEBS Lett. 346, 217-220[CrossRef][Medline] [Order article via Infotrieve] |
20. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
21. |
Vihinen, H.,
and Saarinen, J.
(2000)
J. Biol. Chem.
275,
27775-27783 |
22. | Daròs, J.-A., and Carrington, J. (1997) Virology 237, 327-336[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sun, G., and Budde, R. J. A. (1999) Biochemistry 38, 5659-5665[CrossRef][Medline] [Order article via Infotrieve] |
24. | Beeler, T., Bruce, K., and Dunn, T. (1997) Biochim. Biophys. Acta 1323, 310-318[Medline] [Order article via Infotrieve] |
25. | Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146[CrossRef][Medline] [Order article via Infotrieve] |
26. | Zaworski, P. G., and Gill, G. S. (1988) Anal. Biochem. 173, 440-444[Medline] [Order article via Infotrieve] |
27. | Puurand, U., Mäkinen, K., Baumann, M., and Saarma, M. (1992) Virus Res. 23, 99-105[Medline] [Order article via Infotrieve] |
28. | Shukla, D. D., Ward, C. W., and Brunt, A. A. (1994) The Potyviridae , pp. 124-125, CAB International, Cambridge, United Kingdom |
29. | Ghoshroy, S., Freedman, K., Lartey, R., and Citovsky, V. (1998) Plant J. 13, 591-602[CrossRef][Medline] [Order article via Infotrieve] |
30. | Citovsky, V., Ghoshroy, S., Tsui, F., and Klessig, D. (1998) Plant J. 16, 13-20[CrossRef][Medline] [Order article via Infotrieve] |
31. | Citovsky, V., Knorr, D., Schuster, G., and Zambryski, P. (1990) Cell 60, 637-647[Medline] [Order article via Infotrieve] |
32. |
Más, P.,
and Beachy, R. N.
(1999)
J. Cell Biol.
147,
945-958 |
33. | Citovsky, V., and Zambryski, P. (1991) Bioessays 13, 373-379[Medline] [Order article via Infotrieve] |
34. |
Citovsky, V.,
Wong, M. L.,
Shaw, A. L.,
Prasad, B. V.,
and Zambryski, P.
(1992)
Plant Cell
4,
397-411 |
35. | Karpova, O. V., Ivanov, K. I., Rodionova, N. P., Dorokhov, Yu. L., and Atabekov, J. G. (1997) Virology 230, 11-21[CrossRef][Medline] [Order article via Infotrieve] |
36. | Dolja, V. V., Haldeman, R., Robertson, N. L., Dougherty, W. G., and Carrington, J. C. (1994) EMBO J. 13, 1482-1491[Abstract] |
37. | Kragler, F., Monzer, J., Shash, K., Xoconostle-Cázares, B., and Lucas, W. J. (1998) Plant J. 15, 367-381[CrossRef] |
38. | Dorokhov, Yu. L., Mäkinen, K., Frolova, O. Yu., Merits, A., Saarinen, J., Kalkkinen, N., Atabekov, J. G., and Saarma, M. (1999) FEBS Lett. 461, 223-228[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Chen, M.-H.,
Sheng, J.,
Hind, G.,
Handa, A. K.,
and Citovsky, V.
(2000)
EMBO J.
19,
913-920 |
40. | Oparka, K. J., Roberts, A. G., Boevink, P., Santa Cruz, S., Roberts, I., Pradel, K. S., Imlau, A., Kotlizky, G., Sauer, N., and Epel, B. (1999) Cell 97, 743-754[Medline] [Order article via Infotrieve] |