INTRODUCTION

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The herpes simplex virus type 1 and type 2 (HSV-1 and -2)¹ ribonucleotide reductases, which convert ribonucleoside diphosphates to the corresponding deoxyribonucleotides, play a key role in the synthesis of viral DNA (1). A peculiar feature of the HSV-1 and HSV-2 ribonucleotide reductases was found in the amino acid sequence of their R1 proteins; in contrast to the R1 of other species, including those of other herpesviruses, the HSV-1 and -2 R1 subunits possess an N-terminal extension of about 350 amino acids (2, 3). It has been clearly shown that this extension, which appears to be linked to the reductase domain by a protease-sensitive region, is dispensable for ribonucleotide reduction (4-7).

From sequence comparisons with eukaryotic PKs, Chung et al. (8) were the first to propose that the unique N-terminal domain of HSV R1 could be a PK domain. Among the experimental evidence that has been accumulated thereafter in favor of this hypothesis, the more convincing are the following: (i) the N terminus of the HSV-2 R1 produced with a bacterial expression system and purified by immunoprecipitation is able to phosphorylate histones and calmodulin (9); (ii) both HSV-1 and -2 R1 are labeled by the ATP analogue [¹⁴C]FSBA, which covalently binds to the active-site lysine of eukaryotic PKs (10, 11); (iii) both HSV-1 and -2 R1 produced in eukaryotic cells retrieve their capacity to be phosphorylated after migration on a denaturing polyacrylamide gel and renaturation on blot (11, 12); (iv) a protein exhibiting a weak homology with the N-terminal domain of HSV-2 R1 (termed FAST) was described as a PK involved in the phosphorylation of TIA-1 during Fas-induced apoptosis (13).

However, subsequent observations indicated that the R1 N-terminal domain should belong to a novel type of PK. Deletions of different parts of the protein showed that several of the classical PK consensus sequences could be removed without loss of protein phosphorylation (10, 11). [¹⁴C]FSBA, which inhibits the R1 labeling by [-³²P]ATP, does not bind to residues located in the putative PK domain but to a site in the reductase domain (10). Phosphorylation of histones observed with the HSV-1 and -2 R1 proteins purified following expression in Escherichia coli was shown to be the result of a contaminant prokaryotic kinase; surprisingly, this kinase appears to be able to phosphorylate, in the presence of MnCl₂, several other eukaryotic proteins. After elimination of the prokaryotic kinase responsible for the histone phosphorylation, both types of R1 retain a weak capacity to be phosphorylated in the presence of MnCl₂ (1 R1 molecule/2,400 being labeled in 30 min). From these observations, even if the rate of ³²P_i incorporation was very low, it was concluded that these proteins had intrinsic PK activity (10, 14). However, in the above mentioned studies, the possibility that the R1 phosphorylation was accomplished by residual contaminant PK(s) was never completely ruled out.

During the development of E. coli and adenovirus expression vectors for the production of HSV-2 R1, we observed that efficient phosphorylation of R1 protein and of casein was obtained only with preparations purified from eukaryotic cells. As improvement in the purification of the HSV-2 R1 expressed in eukaryotic cells greatly diminished the capacity of the protein to incorporate [-³²P]ATP, we critically reanalyzed the origin(s) of R1 phosphorylation for both recombinant proteins. We found that the PK activity attributed to R1 was due to contaminating cellular PKs.

	EXPERIMENTAL PROCEDURES

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Reagents-- Casein , calf thymus histones, calmodulin, heparin, polylysine, and protamine sulfate were from Sigma. [-³²P]ATP (4,500 Ci/mmol) and [-³²P]ATP (4,000 Ci/mmol) were from ICN and [³H]CDP (18 Ci/mmol) from Amersham Life Science. Affi-Prep 10 support, bovine serum albumin standard, and the Bio-Rad protein assay kit were from Bio-Rad. Polyclonal antibodies directed against a peptide corresponding to residues 70-91 of the CKII  and CKII purified from sea urchin were kindly provided by S. Pelech (15). Pure human recombinant CKII was from Boehringer Mannheim. Purified HSV-1 R1 (DN247) was kindly provided by Joe Conner (14).

R1 Purification-- The recombinant adenovirus Ad5BM5-R1 was used to produce BM5-R1 in human 293S cells as described previously (16). Crude cytoplasmic extracts of infected cells were prepared by Dounce homogenization of phosphate-buffered saline washed cells suspended in ice-cold buffer A (50 mM Hepes pH 7.6, 2 mM DTT) followed by a 12,000 × g centrifugation. The resulting supernatant (S₁₂) was clarified by centrifugation at 100,000 × g for 1 h at 4 °C (S₁₀₀). The production and partial purification of the HSV-2 R1 in E. coli with a pET vector (pET-R1) were performed as described by Furlong et al. (17) with the exception that the proteins were precipitated by 25% ammonium sulfate instead of 45%.

Ribonucleotide Reductase Assay-- For assays with the unpurified recombinant subunits, aliquots of the S₁₂-, S₁₀₀-, or ammonium sulfate- treated fractions were centrifuged through Sephadex G-25 columns to remove molecules inhibitory for reductase activity. R1 specific activity was determined by adding to limiting amounts of R1 excess amounts of R2 purified from E. coli bearing the pET-R2 expression vector (60 units/mg). R2 specific activity was determined by adding to limiting amounts of R2 excess amounts of purified R1 obtained from Ad5BM5-R1-infected 293 cells (55 units/mg) (16). The host cell (human or E. coli) ribonucleotide reductase activities were undetectable with the standard assay for the HSV enzyme, which contained 50 mM Hepes, pH 7.8, 50 mM DTT, 50 µM CDP, and 0.25 µCi of [³H]CDP in a volume of 30 µl. One unit was defined as the amount of enzyme generating 1 nmol of dCDP/min (16).

Phosphorylation Assays-- The standard assay to measure the phosphorylation of R1 or other proteins in solution contained in 30 µl, 50 mM Hepes (pH 7.8), 2 mM DTT, 0.3 M NaCl, 5 mM MgCl₂, 10 µM ATP, and 5 µCi of [-³²P]ATP (18). For the phosphorylation of casein , pET-R2, calf thymus histones, 10 µg of each protein were included in the assay whereas for calmodulin 5 µg were used. In those conditions, the rates of phosphorylation were usually linear during the entire 10-min incubation period at 37 °C. In some cases, the reaction mixture contained 25 mM HEPES, pH 7.6, 250 mM NaCl, 1 mM MnCl₂, and 10 µCi of [-³²P]ATP as described by Cooper et al. (10) or 20 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, and 10 µCi of [-³²P]ATP as described by Peng et al. (20). For the glycerol gradient fractions, 20-µl samples were added to the phosphorylation mixture. After Coomassie Blue staining, the reaction was stopped by boiling in gel loading buffer and the proteins were separated by SDS-PAGE. The gel was stained with Coomassie Blue and, when appropriate, the amount of R1 in each lane was evaluated by laser densitometric comparison with R1 standard. To quantify the phosphate incorporation, the bands corresponding to R1 or to other substrate were cut and counted by liquid scintillation spectrometry. ³²P_i incorporation from [-³²P]ATP after immobilization of the proteins on polyvinyl fluoride membrane was performed, unless otherwise stated under "Results," as described by Luo and Aurelian (11). Phosphoamino acid analysis of ³²P-labeled proteins was performed after 1 h of digestion in 6 M HCl at 110 °C (21) whereas a sensitive high performance liquid chromatography method was used to quantify the amount of phosphoamino acids in unlabeled proteins (22).

NDPK and Nucleotide Phosphatase Assays-- NDPK assay conditions were the same as for the ribonucleotide reductase assay except that the reactions, which contained 5 mM ATP and 5 mM MgCl₂, were performed in the absence of HSV-2 R2 subunit. After 30 min at 37 °C, the reaction was stopped by boiling for 10 min and the protein precipitates were removed by centrifugation. Before spotting on polyethyleneimine-cellulose plates, rC, CMP, CDP, and CTP, each at a final concentration of 5 mM, were added as markers. The nucleotides were separated by ascending chromatography with 0.2 M sodium bicarbonate, and the radioactivity associated with each UV-visualized spot was measured. Nucleotide phosphatase measurements were performed in parallel reactions devoid of ATP and MgCl₂ using [³H]CDP as substrate.

Ultrafiltration Binding Assay-- The assay was devised from a method originally described to measure nucleotide binding to the E. coli R1 (23). Briefly, purified R1 protein was incubated for 15 min at 37 °C usually at a concentration of 2 µM with 0-10 µM [-³²P]ATP in 150 µl of the phosphorylation assay mixture containing either 5 mM MgCl₂ or 1 mM MnCl₂. Ultrafiltration used to measure free ATP concentration was performed as we recently described (19). The specific binding of the nucleotide to the R1 was calculated by subtracting the nonspecific binding measured in the absence of R1.

	RESULTS

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HSV-2 R1 Produced in E. coli Does Not Efficiently Incorporate [-³²P]ATP-- To study both domains of the HSV-2 R1, the R1 gene was introduced in the adenovirus Ad5E1E3 to create the recombinant Ad5BM5-R1 (16), and in the bacterial expression vector pET11c, which gave pET-R1. Purification of both types of recombinant R1 by peptidoaffinity gave protein preparations, which contained <5% of host cell protein contaminants (Fig. 1). Initial phosphorylation assays were performed with MgCl₂ using conditions optimized for HSV-1 R1 purified from HSV-1-infected cells (18). Surprisingly, whereas the reductase activity of BM5-R1 and pET-R1 was similar (Table I), phosphorylation of the R1 protein and casein could be observed only with preparations purified from eukaryotic cells (Fig. 2, lanes 1-4). Phosphoamino acid analysis of the ³²P-labeled BM5-R1 detected phosphorylation on serine and threonine residues. In addition, we noticed that the rate of BM5-R1 phosphorylation among different batches of purified protein varied largely from 0.04 to 0.64 nmol/min/mg with a parallel variation in the extent of the phosphorylation of either casein (from 0.01 to 0.17 nmol/min/mg) or other substrates. Histones were 32-fold poorer phosphate acceptors than casein. Calmodulin was also efficiently phosphorylated but only when assayed in the presence of 10 µM polylysine. IgGs (either polyclonal or monoclonal) and the recombinant R2 subunit of HSV-2 ribonucleotide reductase were not phosphorylated at all. Interestingly, the formation of the ribonucleotide reductase holoenzyme by the addition of R2 did not alter the rate of R1 phosphorylation.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Peptidoaffinity purification of recombinant HSV-2 R1. Analysis by SDS-PAGE and Coomassie Blue staining of various fractions of BM5-R1 (lanes 1-5) and pET-R1 (lane 6) purification. Lane 7, 4 µg of purified HSV-1 R1 (DN247).

View this table: [in this window] [in a new window]   Table I Comparison of enzymatic activities at different stages of HSV-2 R1 purification BM5-R1 and pET-R1 proteins were purified by peptidoaffinity with (+) or without () a NaCl wash during the R1 binding to the column. Enzymatic activities were measured as described under "Experimental Procedures."

View larger version (66K): [in this window] [in a new window]   Fig. 2.   In vitro phosphorylation of recombinant HSV-2 R1. Analysis of 32P-labeled proteins by SDS-PAGE followed by autoradiography after incubation in the standard conditions described under "Experimental Procedures." Left panel, comparison of BM5-R1 (2 µg, lanes 1 and 2) and pET-R1 (2 µg, lanes 3 and 4) labeling in the absence (lanes 1 and 3) or in the presence of 10 µg of casein  (lanes 2 and 4). Middle panel, BM5-R1 labeling after different steps of purification using a similar amount (2 µg) of R1 in each case: lane 5, S100; lane 6, R1 from a standard purification; lane 7, R1 from a purification with a 2 M NaCl wash; lane 8, same R1 as in lane 7 plus 1/10 of the NaCl wash. Right panel, pET-R1 (1 µg, lane 10) labeling produced by unwashed BM5-R1 (0.1 µg); lane 9, BM5-R1 control.

View larger version (66K): [in this window] [in a new window]   Fig. 3.   Phosphorylation of recombinant HSV-2 R1 in standard and blot assays. Left panel, analysis of 32P-labeled proteins as described in Fig. 2. Lane 1, 20 µg of an extract of Ad5E1E3-infected 293 cells; lanes 2-4 contain 2 µg of pET-R1 either alone (lane 2), or with 20 µg of the lane 1 extract (lane 3), or 0.5 unit of CKII (lane 4). Right panel, purified BM5-R1 (5 µg, lane 5), extract of Ad5E1E3-infected cells (50 µg, lane 6), same extract as in lane 6 plus 5 µg of pET-R1 (lane 7), and 5 µg of pET-R1(lane 8) were subjected to SDS-PAGE and blotted. After denaturation and renaturation of the proteins, the membrane was incubated in the blot assay mixture, washed, and dried before autoradiography.

A High Ionic Wash Decreased the BM5-R1 Phosphorylation-- To demonstrate that a PK activity was co-purifying with R1, we attempted to separate the enzyme from its substrate by washing with 2 M NaCl the BM5-R1 protein bound to the affinity column. As shown in Fig. 2, this wash produced a 40-fold reduction in the R1 (compare lanes 6 and 7) and casein phosphorylation (Table I). Calmodulin and histone phosphorylations were no longer detectable (data not shown). The addition of a small fraction of the NaCl wash desalted and concentrated by ultrafiltration restored a substantial level of R1 labeling (lane 8). The absence of R1 in the NaCl wash, shown in Fig. 1 (lane 4) with a Coomassie Blue-stained gel, was clearly demonstrated by a silver-stained gel and a ribonucleotide reductase assay (data not shown). In Table I, the capacity of the protein to incorporate ³²P_i after each step of purification is compared with its reductase activity. In addition, the activity of NDPK, a highly active cellular enzyme (650 µmol/min/mg; see Refs. 25 and 26), is also presented. Whereas the specific activity of ribonucleotide reductase increased 10-fold in parallel with the concentration of the R1 subunit indicating that the purification procedure did not affect the subunit structure, the R1 phosphorylation exhibited an overall decrease of 300-fold. The observation that the decrease was much less significant after the affinity step than after the NaCl wash (8-fold compared with 40-fold) is also an indication that a co-purifying PK could be responsible of the R1 labeling. The more efficient removal for NDPK by the affinity step (30-fold compared with 5-fold) strengthens this hypothesis. It is noticeable that the residual NDPK activity is 1,000-fold greater than the rate of R1 phosphorylation. Nevertheless, it would be difficult to conclude that this activity is catalyzed by the R1 protein itself because, once again, pET-R1 did not exhibit such an activity (<1 pmol/min/mg). Thus, it is far more plausible to estimate, from the known specific activity of human NDPK, that our pure BM5-R1 preparations contain a 0.0002% level of NDPK contamination than to conclude that the HSV-2 R1 is a primitive NDPK exhibiting 8 × 10⁶ less activity than the human enzyme.

R1 Phosphorylation on Blot Is Produced by a Movable PK-- The HSV2-R1 labeling seen in PK assays on blot has been taken as a nearly irrefutable proof for the autophosphorylation potential of this protein because, in such an assay, the denatured proteins are separated by SDS-PAGE before their blotting onto a membrane (11). Two hypotheses were successively considered to explain how a contaminating eukaryotic PK could be responsible for the R1 labeling on the blot. First, comigration of the contaminating PK with the R1 protein on the gel appears likely from initial observations made in PK assays on blot similar to the one presented in the right panel of Fig. 3. As can be seen, BM5-R1 from preparations unwashed with NaCl exhibited a high degree of phosphorylation (lane 5), whereas pET-R1 was not labeled (lane 8). A faint labeled band was seen at the R1 position in an adjacent lane, which contained a crude extract of Ad5E1E3-infected 293 cells. To test if this comigrating protein could be responsible for the R1 labeling, an aliquot of the Ad5E1E3 extract was mixed with pET-R1 in gel loading buffer and immediately boiled to prevent protein phosphorylation prior to gel electrophoresis. The added extract did not produce any increase in the signal seen at the R1 position (lane 7), making it unlikely that the protein involved in the R1 labeling was comigrating on the gel. In a subsequent experiment (Fig. 4), very puzzling results were obtained for R1 preparations at different stages of purification; whereas in the tube assays the extent of labeling corresponded to the degree of R1 purification, in the blot assay the most purified protein (lane 1) incorporated as much ³²P_i as the less purified R1 (lane 2). These results led us to explore as second alternative that the R1 labeling on blot was produced by a PK that is able to move from its location on the membrane and to interact with R1 during the PK assay.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of recombinant BM5-R1 in blot assays is produced by a movable PK. Left panel, purified BM5-R1 washed with 2 M NaCl (5 µg; lane 1), purified BM5-R1 unwashed with NaCl (5 µg, lane 2), same R1 as in lane 1 plus 1/10 of the NaCl wash (lane 3), and same R1 as in lane 1 plus 50 µg of Ad5E1E3-infected cell extract (lane 4), were subjected to PK assay on blot as described in Fig. 3. Middle panel, duplicate aliquots of proteins of the left panel were blotted, but instead of being further processed as a whole piece, the membrane was cut in four strips corresponding to each lane. Thereafter, each piece of membrane was treated in individual container. Right panel, a third series of the same protein aliquots were blotted, and the membrane was cut in small squares around the R1, which were further processed individually.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Localization of the movable PK in the blot assay. Panel A, a strip of membrane corresponding to 50 µg of Ad5E1E3-infected cell extract was cut into 10 1-cm pieces, which were each individually incubated with pieces of membrane cut around 10 BM5-R1 (5 µg) bands. The diagram illustrates how the pairs of pieces were placed for the autoradiography after the blot assay. Panel B, human recombinant CKII (2 units) was blotted with 5 µg of BM5-R1 in an adjacent lane. Before the denaturation step, the membrane was cut in a piece containing the CKII lane and only the R1 area for the R1 lane. The autoradiogram was cut to show the area corresponding exactly to the membrane.

View larger version (67K): [in this window] [in a new window]   Fig. 6.   Phosphorylation by CKII is necessary for CKII-R1 interaction during the blot assay. Left panel, a membrane excision in a gel lane containing 50 µg of Ad5E1E3 extract, which eliminated proteins between 35 and 45 kDa, abolished labeling of the adjacent BM5-R1 (5 µg) in a blot assay. The labeling background showed the contour of the piece of membrane. BM5-R1 labeling was also abolished by a smaller deletion (40-44 kDa) (data not shown). Middle panel, deletion of proteins below 30 kDa for a lane of pure human CKII did not affect labeling of the adjacent BM5-R1 (5 µg). Right panel, a membrane containing 50 µg of Ad5E1E3 extract (lane 1), 2 µg of pET-R1 phosphorylated with unlabeled ATP and 4 units of CKII for 90 min in solution (lane 2), 2 µg of unphosphorylated pET-R1 (lane 3), and 2 µg of BM5-R1 was submitted to a blot assay. The strong labeling obtained with the phosphorylated pET-R1 was reproduced in a second experiment where only the two central lanes were duplicated.

CKII Co-purifies with R1-- Several observations suggested that CKII could be the main PK co-purifying with R1, which phosphorylated R1 on blot. (i) The catalytic  subunit of the human CKII has a M_r of 42,000. (ii) The N-terminal domain of R1 possesses several potential phosphorylation sites for CKII. (iii) This PK has been reported to co-purify with a large number of diverse proteins. Using pure CKII from two sources (sea urchin and human recombinant), we observed that pET-R1 was indeed a good substrate for this PK (Fig. 3, lane 4). Similar K_m values of 6 and 7 µM were measured for ATP in the presence of 5 mM MgCl₂ for the phosphorylation of BM5-R1 by its co-purifying PK and that of pET-R1 by CKII, respectively. A hallmark of CKII is that GTP can serve nearly as well as ATP as the phosphate donor. We found that GTP inhibited similarly the phosphorylation of pET-R1 by CKII and that of BM5-R1 by its co-purifying PK (Fig. 7A). These results indicated that CKII could be the major PK co-purifying with R1. This was substantiated by the obtainment of similar inhibition curves with heparin (Fig. 7B), a well known potent inhibitor of CKII. Furthermore, polyclonal antibodies against the CKII catalytic subunit detected on different preparations of BM5-R1, a 44-kDa protein, in amounts that correlated well with the levels of R1 phosphorylation measured in our standard PK assay (Fig. 7C, lanes 2 and 3). We were unable to detect CKII  in the NaCl-washed preparation (lane 1); from a value of 5 µmol/min/mg for the CKII specific activity (27), the 10 µg of protein loaded on the gel should contained about 2 pg of CKII , an amount that falls 5-fold below the limit of detection of our immunoblot assay.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   The BM5-R1 co-purifying PK shows CKII-like properties. A, effect of GTP on R1 phosphorylation. B, effect of heparin on R1 phosphorylation. Phosphorylation was assayed in standard conditions in the presence of casein  with either 2 µg of BM5-R1 unwashed with NaCl () or with 2 µg of pET-R1 and 2 units of human CKII (). C, detection of the 44-kDa CKII  protein by immunoblotting with anti-peptide (residues 70-91 of human CKII ) polyclonal antibodies in two BM5-R1 preparations purified by the standard procedure exhibiting medium (40 pmol/min/mg) (lane 2, 5.0 µg) or high (230 pmol/min/mg) (lane 3, 2.5 µg) rate of phosphorylation in our standard conditions. CKII  was not detected in purified BM5-R1 washed with 2 M NaCl (lane 1, 10.0 µg) nor in purified pET-R1(lane 4, 2.5 µg). Human recombinant CKII holoenzyme (1 ng) was added in lane 5.

Identification of CKII as the PK Responsible for the R1 Labeling on Blot-- By doing an experiment similar to the one shown in Fig. 5A for the localization of the movable PK, we demonstrated that the pure recombinant CKII was able to move in the PK assay on blot and to give a strong signal at the BM5-R1 position (Fig. 5B). To verify that this labeling represented R1 phosphorylation and not autophosphorylated CKII bound to R1, we eluted the labeled protein from the membrane and resubmitted it to SDS-PAGE. All the radioactivity migrated at the R1 position showing that CKII autophosphorylation did not contribute significantly to the radioactive signal seen in those experiments. Furthermore, by deleting different parts of lanes containing samples of crude extract as illustrated in the left and middle panels of Fig. 6, we found that only proteins having M_r between 40,000 and 44,000 were necessary to produce R1 labeling on blot. The deletion removing proteins below 30,000 that did not affect the level of labeling clearly showed that the regulatory  subunit of CKII (M_r, 26,000) was unessential to R1 phosphorylation by the CKII .

Elimination of pET-R1 and NaCl-washed BM5-R1 Phosphorylation by Velocity Sedimentation-- To further examine the origin of the low level of phosphorylation either of pET-R1 in the presence of MnCl₂ (2 × 10⁷ mol of ³²P/min/mol) or of the NaCl-washed BM5-R1 preparation, both proteins were submitted to glycerol gradient centrifugation in 250 mM NaCl. Following this procedure, pET-R1 (Fig. 8A) and histones (not shown) were no longer phosphorylated using the conditions described by Cooper et al. (10), suggesting separation of the contaminating PK(s). Unfortunately, we were unable to localize in which fraction(s) the kinase had sedimented, but the possibility that the loss of phosphorylation could be due to R1 inactivation was excluded by the observation that the sedimentation procedure caused only a slight reduction of the R1 reductase activity. Analysis of the BM5-R1 gradient revealed a nearly complete separation of the residual co-purifying kinase CKII, which was easily detected by challenge with casein; an estimate of the molecular mass of the PK responsible for maximal casein phosphorylation gave 110,000, which corresponds well to that of the 125-kDa CKII holoenzyme (Fig. 8B). The trace amount of BM5-R1 phosphorylation, which with our standard conditions occurred in fraction 5 at a rate of 40 fmol/min/mg, was sensitive to heparin (Fig. 8B, inset) and GTP inhibition (not shown) indicating that it is most probably due to residual CKII (1 CKII mol/10⁸ R1 mol). Assays performed with conditions described by Cooper et al. (10) or by Peng et al. (2 mM MnCl₂ and 5 mM MgCl₂; Ref. 20) did not increase the rate of the reaction.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Elimination of R1 phosphorylation by glycerol gradient centrifugation. pET-R1 (panel A) and BM5-R1 washed with 2 M NaCl (panel B) were subjected to velocity sedimentation or, for controls, incubated in 30% glycerol as described under "Experimental Procedures." Samples of 20 µl were assayed using the conditions described by Cooper et al. (10) for the phosphorylation of pET-R1 (), or using the standard conditions for the phosphorylation of BM5-R1 () and casein (), and the amount of R1 protein (if detectable, >0.05 µg) was quantified by Coomassie Blue staining (). Inset, the BM5-R1 phosphorylation () was also measured in presence of heparin (×). Closed symbols on the x axis represent the phosphorylation obtained with control samples: 2 µg for pET-R1 and 1 µg for BM5-R1. The arrow indicates the position of catalase.

	DISCUSSION

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From studies done on limited amounts of purified R1 obtained from HSV-2-infected cells, we and others had proposed that this protein possessed an intrinsic PK activity (18). When the HSV-2 R1 became available in larger amounts as a recombinant protein produced either in adenovirus-infected cells or in E. coli, we searched to totally exclude the involvement of contaminating PKs before undertaking a detailed analysis of this unusual PK. The results of these works presented here suggest that the HSV-2 R1 protein does not by itself possess a PK activity but is instead a good substrate for host cell PKs. This conclusion is based mainly on the finding that an extensive purification of both types of recombinant protein led to a parallel decrease of their putative autophosphorylation potential and their putative capacity to phosphorylate exogenous substrates. As the ribonucleotide reductase activity of the R1 protein was not altered by the purification procedure, the disappearance of these enzymatic phosphorylations is not attributable to R1 instability. Moreover, the finding that the protein does not significantly bind ATP, which is strong evidence against an intrinsic PK activity, also argues against any other intrinsic enzymatic activity involving ATP as phosphate donor.

The most likely explanation for the weak phosphorylation seen with our recombinant protein produced in E. coli is that it contains a trace amount of one or several bacterial PK(s). This is supported by the complete elimination of phosphorylation by glycerol gradient centrifugation. Unfortunately, we have been unable to identify any E. coli PK involved in the pET-R1 labeling probably because it was present in too low of an amount. With a method of purification that differs markedly from ours, others have obtained higher levels of phosphorylation for either the full-length HSV-1 or HSV-2 R1 subunits or for a truncated HSV-2 R1 consisting of amino acids 1 to 270 (10, 20). In an attempt to explain these differences, we have purified by peptidoaffinity the recombinant HSV-1 R1 from crude bacterial extracts kindly provided to us by J. Conner. This purified protein exhibited a level of phosphorylation (5 fmol/min/mg) similar to the one obtained with pET-R1 (2 fmol/min/mg), suggesting that our method of purification is more effective to eliminate bacterial PK(s).² We considered it unlikely that the autophosphorylation potential of pET-R1 would have been inactivated following efficient in vivo autophosphorylation because we were unable to detect the presence of phosphate on this protein using a high performance liquid chromatography method that detected 1.8 mol of phosphate/mol of maximally phosphorylated BM5-R1.

The purification by our standard peptidoaffinity method of either the HSV-2 R1 from recombinant adenovirus-infected cells described here or of the HSV-1 R1 from HSV-infected cells (18) yielded proteins that were phosphorylated at rates previously observed for the autophosphorylation of several PKs. In addition, the purified HSV-1 R1 appeared to be able to phosphorylate casein at a rate equal to 1/100 of the one of CKII for the same substrate, suggesting that the protein had an intrinsic PK activity (18). However, after additional purification either by a NaCl wash followed by velocity sedimentation on glycerol gradient for BM5-R1 or by immunoprecipitation with R1 specific antibodies for the HSV-1 R1,³ both proteins lost in parallel their putative autophosphorylation potential and their capacity to phosphorylate casein and histones. These observations are more compatible with the hypothesis that contaminant PK(s) had been separated from R1, but the possibility that the more extensive purification has eliminated a factor involved in R1 activation cannot be completely ruled out.

Our search of an explanation for the R1 labeling on blot led to the finding that CKII was co-purifying with BM5-R1. Subsequent analyses strongly suggested that it was the major PK responsible of BM5-R1 labeling in solution. (i) The amount of CKII detected by immunoblot in different R1 preparations correlated well with the level of R1 phosphorylation. (ii) The co-purifying PK exhibited several standard criteria used to distinguish CKII from other serine/threonine PK. (iii) Similar phosphopeptide maps were obtained following trypsin digestion of either pET-R1 phosphorylated by CKII or of two BM5-R1 preparations exhibiting low or high rate of R1 phosphorylation.⁴ Copurification of CKII with a protein substrate is not peculiar to HSV-2 R1; it has been reported for a large number of cytoplasmic or nuclear proteins exhibiting diverse functions (29-38). Some of these CKII substrates, for example DNA topoisomerase II and Grp94, were, like the HSV-R1, reported to possess autophosphorylation potential until improvements in their purification clearly demonstrated their substrate nature (30, 36). Interestingly, it has been shown that proteins containing serine- and glutamic acid-rich cluster of amino acids such as the PK, p130PITSLRE, could upon phosphorylation by CKII bind specifically with Src homology 2 domains in a phosphotyrosine-independent manner (39). We have observed that the HSV-2 R1 exhibited a similar property in in vitro binding assays done with glutathione S-transferase fused Src homology 2 domains.⁵

The most astonishing demonstration of the present work is that phosphorylation of a protein on blot as proof of autophosphorylation could be misleading. Proving intrinsic autophosphorylation has often been a difficult task mainly because eukaryotic PKs exhibit a strong tendency to co-purify with their protein substrates. The PK assay on blot was thought to give irrefutable evidence of the autophosphorylation potential of a protein if care was taken to demonstrate that the ³²P_i was incorporated in the protein of interest and not in the protein used to block the membrane (40). Our finding that a PK can detach from its position on a membrane, bind with high affinity to one of its protein substrates located elsewhere, and phosphorylate it is an artifact that could be easily prevented by the isolation of the putative PK by an appropriate cutting of the membrane. Is this phenomenon a frequent cause of [-³²P]ATP labeling on blot? We observed that the labeling on blot of most of the proteins present in a crude extract was not affected either by the removal of the part of the membrane containing the CKII  subunit or by isolating them on small pieces of membrane. However, a positive PK assay on blot reported for the BCR gene product (41) could be another case of artifactual phosphorylation by CKII. As the HSV R1, the BCR gene product contains potential CKII phosphorylation sites in an area of the protein rich in serine and in acidic amino acids shown to be essential for the protein phosphorylation in solution (41).

CKII is not the only eukaryotic PK that is able to phosphorylate R1. Using pure recombinant PKA, we have recently detected a weak phosphorylation of pET-R1. PKC could be another PK able to modify the R1 protein as five potential phosphorylation sites are detected in the protein by the program Prosite. Using antibodies against these two PKs, we have been unable to detect their presence in any of our preparations after the standard peptidoaffinity step.⁵ However, as for CKII, we cannot exclude the possibility that they are present in amounts below the detection limits of our immunoblots. Our difficulty in obtaining preparations of both types of recombinant R1 that did not exhibit significant level of phosphorylation illustrates how important it is to use purification procedures that effectively separate the protein of interest from co-purifying PK(s). A close examination of all the other studies on R1 phosphorylation revealed that the presence of contaminating PK was never completely ruled out (8, 10, 14, 20, 42-46). In most cases, immunoprecipitation was used to purify the R1 protein. Such a procedure has been used many times to show in vivo interactions between proteins and is well known for its propensity to falsely identify PKs. It is surprising that, in several of these studies, coprecipitation of a great number of proteins with the R1 has been noticed without seriously addressing the possibility that bona fide PKs could be present in the precipitates and responsible for R1 phosphorylation (20, 46). In recent experiments,⁵ we have detected the coprecipitation with HSV-2 R1 of tyrosine PKs of the family of growth factor receptors and of the Src family, and also of CKII and PKC. Others have reported that the HSV-2 R1 possesses Src homology 3 binding sites that could be involved in interactions with tyrosine PKs of the Src family (45). We believe that the tyrosine PKs that we have found in complexes with R1 could have been responsible for the reported phosphorylation of calmodulin and IgGs by the HSV-2 R1 (20). These proteins are well known substrates of tyrosine PKs (47-49). CKII also could have contributed to calmodulin phosphorylation, whereas R2 phosphorylation could be the result of PKA activity, as we have observed that this protein is an in vitro substrate of this enzyme. Unfortunately, the utilization of immunoprecipitation to purify mutated R1 proteins in several studies casts doubts about the value of previous conclusions that certain mutated residues could be important for the autophosphorylation potential of the protein (10, 20, 45, 46). It is important to realize that the level of phosphorylation of a mutated protein such as R1 could be affected by changes in either its affinity for the coprecipitating PKs or conformation-dependent availability of phosphorylation sites. The importance of the latter possibility has been underlined in a recent phosphorylation-related analysis of several mutated forms of p53, where it was shown that the conformation of p53 is of central importance not only for its availability as a substrate for different PKs but also for the phosphorylation pattern generated by the same PK (50).

In conclusion, our present results suggest that the N-terminal domain of HSV-R1 is not a PK domain. However, we cannot completely rule out that an interaction with a cellular factor is necessary to activate the HSV-R1 cryptic PK activity or that it can phosphorylate only certain specific substrates. Even if the N-terminal domain of HSV-R1 is not a PK domain, its capacity to bind with high affinity to cellular proteins could be important in viral pathogenesis. In this respect, it is important to recall that the HSV-R1 is synthesized during the lytic cycle in amounts large enough to produce dominant negative effects (1-2% of total cellular proteins).