From the Institut du Cancer de Montréal and
Centre de Recherche du Centre Hospitalier
de l'Université de Montréal, Pavillon Notre-Dame, 1560 est, Sherbrooke, Montréal, Québec H2L 4M1, Canada, and the
¶ Institut de Recherche en Biotechnologie, 6100 avenue Mont-Royal,
Montréal, Québec H4P 2R2, Canada
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ABSTRACT |
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The N terminus of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is believed to be a protein kinase domain mainly because the R1 protein was phosphorylated in a protein kinase assay on blot. Using Escherichia coli and adenovirus expression vectors to produce R1, we found that, whereas the reductase activity of both recombinant proteins was similar, efficient phosphorylation of R1 and casein in the presence of Mg2+ was obtained only with the R1 purified from eukaryotic cells. Phosphorylation of this R1, in solution or on blot, results mainly from the activity of casein kinase II (CKII), a co-purifying protein kinase. Labeling on blot occurs from CKII leakage off the membrane and its subsequent high affinity binding to in vivo CKII-phosphorylated R1. CKII target sites were mapped to an acidic serine-rich segment of the R1 N terminus. Improvement in purification of the R1 expressed in eukaryotic cells nearly completely abolished its phosphorylation potential. An extremely low level of phosphorylation observed in the presence of Mn2+ with the R1 produced in E. coli was probably due to an unidentified prokaryotic protein kinase. These results provide evidence that the herpes simplex virus type 2 R1 does not possess an intrinsic protein kinase activity.
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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
32Pi 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.
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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%.
The peptidoaffinity method used for the R1 purification was modified from the one developed for R1 expressed in HSV-1-infected cells (18). Briefly, 100-150 mg of S100 proteins were diluted at 2 mg/ml in buffer A containing 2.5 mM bacitracin and loaded on a 40-ml column of Affi-Prep-coupled peptide. After washing with 50 column volumes of buffer A, the R1 protein was eluted with 15 ml of buffer A containing 200 µM peptide acetyl-YAGAIVNDL. In some cases, a high salt wash (40 ml of 2 M NaCl in buffer A) was performed before the R1 elution. Ultrafiltration with Centriprep-30 (Amicon) was used to concentrate the eluted R1 and to reduce the concentration of the eluting peptide below 0.2 µM. The protein purity was assessed by laser densitometric scanning of a lane containing 10 µg of protein on a Coomassie Blue-stained gel and by immunoblot analysis performed with a polyclonal rabbit antiserum raised against the purified protein. Purified preparations of both R1 proteins usually containedRibonucleotide 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. 32Pi
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.
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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 Ad5
E1
E3 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.
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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
32Pi 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
Ad5E1
E3-infected 293 cells. To test if this comigrating protein
could be responsible for the R1 labeling, an aliquot of the
Ad5
E1
E3 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 32Pi 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.
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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
Mr 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 Km
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.
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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 Mr 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
(Mr, 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.
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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 32Pi 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).
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Canada and by the National Research Council of Canada.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.
This is National Council of Canada publication 41402.
§ To whom correspondence should be addressed: Centre de Recherche du Centre Hospitalier de l'Université de Montréal, 1560 est, Sherbrooke, Montréal, Québec H2L 4M1, Canada. Tel.: 514-281-6000 (ext. 6827); Fax: 514-896-4689; E-mail: langeliy{at}ere.umontreal.ca.
Present address: Biochem Therapeutic Inc., Laval, Québec
H7V 4A7, Canada.
** Recipient of a studentship from "Société de Recherche sur le Cancer de Montréal."
Recipient of a scholarship from "Fonds de Recherche en
Santé du Québec."
1
The abbreviations used are: HSV, herpes simplex
virus; PK, protein kinase; FSBA, p-fluorosulfonylbenzoyl
5-adenosine; CKII, casein kinase II; DTT, dithiothreitol; NDPK,
nucleoside diphosphate kinase; PAGE, polyacrylamide gel
electrophoresis.
2 L. Champoux and Y. Langelier, unpublished observations.
3 N. Lamarche and Y. Langelier, unpublished observations.
4 J. Lee and Y. Langelier, unpublished observations.
5 S. Bergeron, C. Guilbault, and Y. Langelier, manuscript in preparation.
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