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Phosphorylation Down-regulates the RNA Binding Function of the Coat Protein of Potato Virus A

2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês

10.1074/jbc.m009551200

1083-351X

Konstantin I. Ivanov, Pietri Puustinen, Andres Merits, Märt Saarma, Kristiina Mäkinen,

Plant and Fungal Interactions Research

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 phosphorylatedin 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. 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 phosphorylatedin 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 (1Kawakami S. Padgett H.S. Hosokawa D. Okada Y. Beachy R.N. Watanabe Y. J. Virol. 1999; 73: 6831-6840Crossref PubMed Google Scholar, 2Karpova O.V. Rodionova N.P. Ivanov K.I. Kozlovsky S.V. Dorokhov Yu. L. Atabekov J.G. Virology. 1999; 261: 20-24Crossref PubMed Scopus (63) Google Scholar, 3Waigmann E. Chen M.-H. Bachmaier R. Ghoshroy S. Citovsky V. EMBO J. 2000; 19: 4875-4884Crossref PubMed Scopus (144) Google Scholar). The functional role of MPs is to assist the spread of viral progeny from cell to cell and over long distances (reviewed in Refs. 4Carrington J.C. Kasschau K.D. Mahajan S.K. Schaad M.C. Plant Cell. 1996; 8: 1669-1681Crossref PubMed Scopus (532) Google Scholar, 5Lazarowitz S.G. Beachy R.N. Plant Cell. 1999; 11: 535-548Crossref PubMed Scopus (319) Google Scholar, 6Santa Cruz S. Trends Microbiol. 1999; 7: 237-241Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 7Lee J.-Y. Yoo B.-C. Lucas W.J. Planta. 2000; 210: 177-187Crossref PubMed Scopus (43) Google Scholar). There is evidence that the 30-kDa MP of tobacco mosaic virus (TMV; genusTobamovirus) is phosphorylated when expressed in insect cells from a baculovirus vector (8Atkins D. Roberts K. Hull R. Prehaud C. Bishop D.H.L. J. Gen. Virol. 1991; 72: 2831-2835Crossref PubMed Scopus (15) Google Scholar), in TMV-infected protoplasts (9Watanabe Y. Ogawa T. Okada Y. FEBS Lett. 1992; 313: 181-184Crossref PubMed Scopus (57) Google Scholar,10Haley A. Hunter T. Kilberstis P. Zimmern D. Plant J. 1995; 8: 715-724Crossref PubMed Scopus (48) Google Scholar), and in the cell wall-enriched fractions of transgenic plants expressing the wild-type MP and its mutants (3Waigmann E. Chen M.-H. Bachmaier R. Ghoshroy S. Citovsky V. EMBO J. 2000; 19: 4875-4884Crossref PubMed Scopus (144) Google Scholar, 11Citovsky V. McLean B.G. Zupan J. Zambryski P. Genes Dev. 1993; 7: 904-910Crossref PubMed Scopus (143) Google Scholar). 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 (12Sokolova M. Prüfer D. Tacke E. Rohde W. FEBS Lett. 1997; 400: 201-205Crossref PubMed Scopus (62) Google Scholar). 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 (13Seron K. Bernasconi L. Allet B. Haenni A.-L. Virology. 1996; 219: 274-278Crossref PubMed Scopus (11) Google Scholar). 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 (11Citovsky V. McLean B.G. Zupan J. Zambryski P. Genes Dev. 1993; 7: 904-910Crossref PubMed Scopus (143) Google Scholar). Proteolytic processing was found to be an alternative mechanism to phosphorylation for inactivation of TMV MP inArabidopsis thaliana (14Hughes R.K. Perbal M.-C. Maule A.J. Hull R. Mol. Plant-Microbe Interact. 1995; 8: 658-665Crossref PubMed Scopus (17) Google Scholar). 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 (1Kawakami S. Padgett H.S. Hosokawa D. Okada Y. Beachy R.N. Watanabe Y. J. Virol. 1999; 73: 6831-6840Crossref PubMed Google Scholar). 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 (2Karpova O.V. Rodionova N.P. Ivanov K.I. Kozlovsky S.V. Dorokhov Yu. L. Atabekov J.G. Virology. 1999; 261: 20-24Crossref PubMed Scopus (63) Google Scholar). 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 (3Waigmann E. Chen M.-H. Bachmaier R. Ghoshroy S. Citovsky V. EMBO J. 2000; 19: 4875-4884Crossref PubMed Scopus (144) Google Scholar), 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. 15Revers F. Le Gall O. Candresse T. Maule A.J. Mol. Plant-Microbe Interact. 1999; 12: 367-376Crossref Scopus (195) Google Scholar). 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. 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. 16Hammond J. Lawson R.H. J. Virol. Methods. 1988; 20: 203-217Crossref PubMed Scopus (48) Google Scholar, 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 (17McDonald J.G. Bancroft J.B. J. Gen. Virol. 1977; 35: 251-263Crossref Scopus (27) Google Scholar) and dialyzed against the same HEPES buffer containing 0.5 m NaCl. 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 msucrose. 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. Generation of expression constructs was described in Refs. 18Merits A. Guo D. Saarma M. J. Gen. Virol. 1998; 79: 3123-3127Crossref PubMed Scopus (61) Google Scholar and 19Ivanov K.I. Ivanov P.A. Timofeeva E.K. Dorokhov Yu. L. Atabekov J.G. FEBS Lett. 1994; 346: 217-220Crossref PubMed Scopus (20) Google Scholar. 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-β-d-galactopyranoside-inducible expression of N-terminal His6 fusion proteins. The purification scheme of bacterially expressed His6PVA MRPs and His6TMV MP was previously described (18Merits A. Guo D. Saarma M. J. Gen. Virol. 1998; 79: 3123-3127Crossref PubMed Scopus (61) Google Scholar, 19Ivanov K.I. Ivanov P.A. Timofeeva E.K. Dorokhov Yu. L. Atabekov J.G. FEBS Lett. 1994; 346: 217-220Crossref PubMed Scopus (20) Google Scholar). 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, 0.1mNa2HPO4/NaH2PO4, 0.01m 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 (18Merits A. Guo D. Saarma M. J. Gen. Virol. 1998; 79: 3123-3127Crossref PubMed Scopus (61) Google Scholar) and TMV MP (19Ivanov K.I. Ivanov P.A. Timofeeva E.K. Dorokhov Yu. L. Atabekov J.G. FEBS Lett. 1994; 346: 217-220Crossref PubMed Scopus (20) Google Scholar). In vitro transcription reactions were performed as described in Ref. 18Merits A. Guo D. Saarma M. J. Gen. Virol. 1998; 79: 3123-3127Crossref PubMed Scopus (61) Google Scholar.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). 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. 20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar. 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). 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−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. Phosphorylation was measured as the incorporation of radioactivity from [γ-33P]ATP into the purified substrate proteins. Redivue [γ-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[γ-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 [γ-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. 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 mmdithiothreitol, and 20 mm Tris-HCl, pH 7.5, for 20 min at 37 °C. Control reactions in the same buffer with 1 μmokadaic 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 was carried out as described in Ref. 21Vihinen H. Saarinen J. J. Biol. Chem. 2000; 275: 27775-27783Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar. The RNA-protein binding assays were performed according to the method described in Ref. 22Daròs J.-A. Carrington J. Virology. 1997; 237: 327-336Crossref PubMed Scopus (48) Google Scholar 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. Prior to RNA-protein binding assays, in vitrophosphorylation 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 mmTris-HCl, pH 8.0, 1 mm dithiothreitol, 50 mmNaCl, 1 mg ml−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. 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.23Sun G. Budde R.J.A. Biochemistry. 1999; 38: 5659-5665Crossref PubMed Scopus (52) Google Scholar. The four MRPs of PVA (CIP, CP, HC-Pro, and VPg) and the MP of TMV were expressed inEscherichia 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 [γ-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 [γ-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). 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. 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 [γ-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. 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 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 (1Kawakami S. Padgett H.S. Hosokawa D. Okada Y. Beachy R.N. Watanabe Y. J. Virol. 1999; 73: 6831-6840Crossref PubMed Google Scholar, 10Haley A. Hunter T. Kilberstis P. Zimmern D. Plant J. 1995; 8: 715-724Crossref PubMed Scopus (48) Google Scholar, 11Citovsky V. McLean B.G. Zupan J. Zambryski P. Genes Dev. 1993; 7: 904-910Crossref PubMed Scopus (143) Google Scholar). 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 μmokadaic acid). A separate assay was performed with TMV pMP, which wa