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The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli

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The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli
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   Journal of General Virology  (1999),  80 , 1471–1476. Printed in Great Britain ..........................................................................................................................................................................................................  SHORT COMMUNICATION The unique N terminus of herpes simplex virus type 1ribonucleotide reductase large subunit is phosphorylatedby casein kinase 2, which may have a homologue in Escherichia coli  Joe Conner School of Biological and Biomedical Sciences, Department of Biological Sciences, Glasgow Caledonian University,Cowcaddens Road, Glasgow G4 0BA, UK  Studies were performed to determine if the uniqueN-terminal domain of the R1 subunit from herpessimplexvirus(HSV)type1ribonucleotidereductaseis a substrate for casein kinase 2 (CK2). Trans-phosphorylation assays demonstrated that R1 washighlyphosphorylatedbythisenzymewithmultiplephosphorylation sites mapped to the N terminusbetween residues 1 and 245. Immunoprecipitationpull-down assays using R1-specific antisera failed to demonstrate a stable interaction between R1and CK2 but residual amounts of CK2 present afterimmunoprecipitation efficiently transphosphoryl-ated R1. Activity assays with a peptide substrateidentified CK2 in R1 immunoprecipitated frominfected-cell extracts but did not detect activity inR1 proteins immunoprecipitated from bacterialextracts. However, Western blotting identified po- tential  E. coli   homologues of the CK2 alpha andbeta subunits. These results support conclusions that the N-terminal domain of HSV R1 is not aprotein kinase and that all previous results can beexplained by contaminating kinases, principallyCK2. Conversionofribonucleotidestothecorrespondingdeoxy-ribonucleotides by ribonucleotide reductase (RR) is essentialfor the  de novo  synthesis of DNA in all living organisms(Reichard, 1993). Many herpesviruses encode their own RRand the active form is a tetramer of homodimeric R1 and R2subunits (reviewed in Conner  et al ., 1994 a ). RR activity is atarget for antiviral chemotherapy and has provided a paradigmtostudypeptideswhichdisruptprotein–proteininteractionsasa route to the development of antiviral drugs (Dutia  et al .,  Author for correspondence:  Joe Conner.Fax   44 141 331 3208. e-mail J. Conner  gcal.ac.uk  1986; Cohen  et al ., 1986; Marcello  et al ., 1994; Liuzzi  et al .,1994). In addition to their involvement in RR activity, the R1subunits of herpes simplex virus (HSV) types 1 and 2 havebeen reported to possess a serine  threonine phosphokinase(PK) activity (R1PK) (Chung  et al ., 1989; Conner  et al ., 1992 a ;Paradis  et al ., 1991; Cooper  et al ., 1995) that is distinct fromconventional eukaryotic kinases. The novel kinase activity isthought to be located within the N-terminal 310 amino acids,a domain which is unique to HSV R1 and is not required forribonucleotide reduction (Conner  et al ., 1992 b , 1993, 1994 b ;Lankinen  et al ., 1993); also, its role in HSV replication  pathogenesis is not yet established. Protein R1 is essential forvirus pathogenicity as evidenced by the avirulence and failureto reactivate from latency of deletion mutants (Cameron  et al .,1988; Jacobson  et al ., 1989; Yamada  et al ., 1991; Heineman &Cohen, 1994; deWind  et al ., 1993) which could reflect eitherthe lack of RR or N-terminal activity or of both.The HSV R1 subunits are synthesized in infected tissueculture cells with immediate-early kinetics, in contrast to R2which is an early protein (Clements  et al ., 1977; Wymer  et al .,1989; Conner  et al ., 1995). During infection the R1 promoteris uniquely regulated: expression is induced by the immediate-early transactivator Vmw65 (VP16) and, intriguingly, byVmw110 (ICP0) (Desai  et al ., 1993), a viral transactivatorrequired for efficient reactivation from latency (Clements &Stow, 1989; Leib  et al ., 1989; Russell  et al ., 1987). Interestingly,the R1 promoter contains functional response elements forcellular activator protein 1 (AP-1) (Wymer  et al ., 1992; Zhu &Aurelian, 1997), a bipartite complex of two cellular immediate-early proteins, c-Fos and c-Jun. HSV-1 R1 is twice as abundantas R2 in infected cells and the intracellular localization of thefree R1 is distinct from that of the active enzyme (Conner  et al .,1995). These data have been used to propose a role for R1additional to its involvement in RR that requires thephosphorylation of viral  cellular proteins during immediate-early times by the putative N-terminal protein kinase (R1PK).However, there are a number of discrepancies relating tothe putative R1PK (see Peng  et al ., 1996) and these haveculminated in a recent proposal by Langelier  et al . (1998) that 0001-5957  1999 SGM  BEHB  J. Conner   J. Conner 1 2 3 4 5 67 8 9 10 11 12 13 14 15 1617 18 19 20dN245R1dC449R1in112R1in343R1 Fig. 1.  Autoradiographs which demonstrate CK2 transphosphorylation of protein 1R1. Bacterially expressed 1R1 wasimmunoprecipitated with antiserum 106 in the absence (lane 2) or presence of 0  5 (lane 3), 1 (lane 4), 2 (lane 5) or 4 (lane6)  µ g CK2. Lane 1 is an immunoprecipitated control bacterial extract that contains no 1R1-related proteins. Lanes 7–16demonstrate the effects of increasing amounts of CK2 on phosphorylation of bacterially expressed and purified 1R1. Samplesare in duplicate: lanes 7 and 8, 0 ng CK2; lanes 9 and 10, 0  9 ng CK2; lanes 11 and 12, 1  8 ng CK2; lanes 13 and 14,3  6 ng CK2; lanes 15 and 16, 7  2 ng CK2. CK2 phosphorylations of bacterially expressed dN245R1, dC449R1, in112R1 andin343R1 immunoprecipitated in the presence of 4  µ g CK2 using antiserum 106 are shown in lanes 17–20 respectively. the R1 subunit is not a PK but is a good substrate for host cellPKs, particularly casein kinase 2 (CK2). The results presentedhere support this observation and strengthen the conclusionthat the N terminus of HSV R1 is not a protein kinase.AnalysisbySDS–PAGEandautoradiographyofbacteriallyexpressed HSV-1 R1 (1R1) immunoprecipitated by antiserum106 from ammonium sulphate fractions as described in Cooper et al . (1995) in the presence of increasing amounts of purified Drosophila CK2indicatedthatR1wasasubstrateforthiskinase(Fig. 1, lanes 1–6). CK2 was added before immunoprecipitationand, after extensive washing, phosphorylation was determinedby addition of 30  µ l of CK2 assay buffer (50 mM Tris–HClbuffer, pH 8  2, 20 mM MgCl  ) with 1 µ l [  P]ATP to theProtein A–Sepharose pellet. After incubation at 25   C for30 min, the pellet was washed once with CK2 assay buffer and  P incorporation was observed by SDS–PAGE and auto-radiography. The levels of 1R1 phosphorylation increasedwith increasing amounts of CK2; Fig. 1, lane 1 shows animmunoprecipitated control bacterial extract and lanes 2–6showphosphorylationof1R1afterimmunoprecipitationin theabsence (lane 2) or presence of 0  5 (lane 3), 1 (lane 4), 2 (lane 5)or 4  µ g (lane 6) CK2. Bacterially expressed and purified 1R1(Conner  et al ., 1993) was readily phosphorylated also by CK2(Fig.1,lanes7–16).Duplicate5  µ g1R1sampleswereincubatedalone or with increasing amounts of CK2; 1R1 ‘auto-phosphorylation’ (lanes 7 and 8) was only observed afterlongerexposures of the gel to autoradiograph film (not shown)whereas lanes 9–16 readily show transphosphorylation by 0  9(lanes 9 and 10), 1  8 (lanes 11 and 12), 3  6 (lanes 13 and 14)and 7  2 ng (lanes 15 and 16) CK2.The majority of CK2 phosphorylation sites were mappedto the N-terminal domain of 1R1. ImmunoprecipitateddN245R1, a polypeptide lacking the first 244 amino acids(Conner  et al ., 1993), was a poor substrate for CK2 (Fig. 1, lane17) whereas the immunoprecipitated, C-terminally truncated1R1 protein, dC449R1, comprising residues 1–448 (Cooper  etal ., 1995), was phosphorylated (Fig. 1, lane 18). Surprisingly,in112R1, a variant on dC449R1 with a four amino acid insertat residue 112 that abrogated R1PK activity (Cooper  et al .,1995), was a poor CK2 substrate (Fig. 1, lane 19) whereasin343R1, a PK ‘active’ variant of dC449R1 with the same fouramino acid insert at residue 343 was phosphorylated (lane 20).The proteins were immunoprecipitated from ammoniumsulphate fractions in the presence of 4  µ g CK2; antiserum 106immunoprecipitates approximately equal amounts of all fourpolypeptides (Cooper  et al ., 1995). Tryptic digestion (Conner et al ., 1992 b ) of CK2-phosphorylated 1R1 generated 87 kDaC-terminal and 33 kDa N-terminal fragments and the majorityof    P was detected in the N-terminal fragment (data notshown).Experiments using immunoprecipitation assays were per-formed to determine whether there was a stable physicalinteraction between R1 and CK2. CK2 was added to 1R1ammonium sulphate fractions and, after immunoprecipitationwith antiserum 106, the immunocomplexed proteins wereanalysedbySDS–PAGE andWestern blottingusing antiserumspecific for the CK2 alpha or beta subunits: no evidence of any interaction was detected (data not shown). Further inter-action studies, using a more sensitive and quantitativephosphorylation assay with a CK2 substrate peptide,RRREEETEEE, were undertaken. Protein 1R1 was immuno-precipitated from bacterial extracts by either antiserum 106 orMAb7689 in the presence of 8  µ g CK2. After extensivewashes, amounts of CK2 in the immunoprecipitates were BEHC  Phosphorylation of HSV R1 by CK2   Phosphorylation of HSV R1 by CK2 Table 1.  Amounts of CK2 pulled down by 1R1 measured using the peptidephosphorylation assay ProteinImmunoprecipitatingantibody*CK2 added( µ g) † CK2 ‘pulleddown’ ( µ g) ‡ % CK2‘pulled down’Molar ratioR1:CK2 § 1R1 106 8 0  037 0  46 13:11R1 7689 8 0  044 0  55 11:1* 1  1  µ g 1R1 is immunoprecipitated from bacterial extracts by these antibodies (Cooper  et al ., 1995). †  CK2 was added to the 1R1 ammonium sulphate fraction prior to immunoprecipitation. ‡  CK2 pulled down was calculated from the levels of peptide phosphorylation using a calibration curve. §  Molecular masses for molar ratio determinations were as follows: 1R1, 140000 kDa; CK2, 65000 kDa. CK2 α CK2 β 1 2 3 4 - 250K - 160K - 105K - 75K - 50K - 35K - 30K - 25K - 15K b)a) 050001000015000200002500030000   c  p  m   p  e  p   t   i   d  e    1   R   1   2   R   1   d   N   2   4   5   R   1  c  o  n   1   7   1   7  -  p  e  p   C   K   2 Fig. 2.  ( a ) Chart demonstrating levels of CK2 peptide phosphorylation activity present in 1R1, 2R1 and dN245R1 proteinsimmunoprecipitated by antiserum 106 from bacterial extracts or 1R1 (17) immunoprecipitated from infected-cell extracts andassayed in the presence (17) or absence (17  pep) of peptide. Con is a control bacterial extract lacking any 1R1-relatedproteins. No exogenous CK2 was added to any of these samples. ( b ) Western blots of ammonium sulphate fractions frombacterial (lanes 2 and 3) or infected-cell (lanes 1 and 4) extracts probed with antisera specific for CK2 alpha (lanes 3 and 4)or beta (lanes 1 and 2). The positions of molecular mass markers are shown on the right-hand side. determined by peptide phosphorylation and the results arepresented in Table 1. Less than 1% of the available CK2 waspulled down suggesting that there is no strong interactionbetween R1 and CK2. Molar ratios of R1:CK2 were calculatedfrom estimates of the amounts of 1R1 and CK2 in immuno-precipitates and, although the resultant values must be treatedwith some caution, the expected 1:1 ratio for a stable R1–CK2complex was not obtained; 8  µ g CK2 is a 15-fold molar excessover immunoprecipitated R1. Despite the lack of interaction,theresidualamountsofCK2presentafterimmunoprecipitationtransphosphorylated 1R1 (see Fig. 1, lanes 3–6)R1 proteins immunoprecipitated from infected cell andbacterial extracts were investigated for CK2 contaminationusing peptide phosphorylation assays (Fig. 2 a ). Levels of peptidephosphorylationweredeterminedinduplicate samplesof 1R1, HSV-2 R1 (2R1) and dN245R1 immunoprecipitated byantiserum 106 from bacterial extracts and in an immuno-precipitated control extract but no CK2 activity was evident;the levels of    P detected were consistent with background forthis assay. Background levels of phosphorylation were higherin 1R1 immunoprecipitated from infected cell extracts but thishigher level of    P incorporation was augmented in thepresence of the CK2 peptide indicating that this samplecontained contaminant CK2 activity; a similar level of phosphorylation wasobtainedwith 1 ng CK2.The ammoniumsulphate fractions from bacterial and infected-cell extracts usedfor 1R1 immunoprecipitation were analysed by Westernblotting with antisera specific for either CK2 alpha or beta (Fig.2 b ) and both alpha (lane 4) and beta (lane 1) subunits weredetected in infected cell extracts. Surprisingly, a band cor- BEHD  J. Conner   J. Conner responding to the CK2 beta subunit was detected in thebacterial extract (lane 2) and, amongst several cross-reactivebands that were detected with the alpha subunit antiserum(lane 3), one corresponded to CK2 alpha.Protein kinase activity was first described for 2R1 byChung  et al . (1989) and for 1R1 by Paradis  et al . (1991). Theirinitial studies used proteins that were either immuno-precipitated or purified from infected-cell extracts. Since thesefirst reports a number of other results, including expression of the ‘active’ R1PK in  E .  coli  and mutagenesis studies (Conner  etal ., 1992 a ; Cooper  et al ., 1995; Luo & Aurelian, 1992; Luo  etal ., 1991), have consistently demonstrated apparent intrinsicautophosphorylation activity that resides within the N-terminal domain despite a lack of conventional eukaryoticprotein kinase motifs. However, there were someinconsistencies concerning R1PK transphosphorylation of eukaryotic protein kinase substrates. Studies have reportedphosphorylation of histones, casein and calmodulin by 1R1and 2R1 (Chung  et al ., 1990; Paradis  et al ., 1991; Luo  et al .,1991). In contrast, Conner  et al . (1992 a ), Cooper  et al .(1995) and Ali (1995) consistently failed to detect trans-phosphorylation of any of these substrates. Indeed, Conner  etal . (1992 a ) and Cooper  et al . (1995) reported a contaminant  E . coli  protein kinase which phosphorylated histones in their R1preparations and these authors recommended caution in theinterpretationofanyR1PKresults.Peng et al .(1996)attemptedto reconcile these inconsistencies by differences in the optimalassayconditionsforthe1R1and2R1PKsbutthediscrepancieshave resulted in speculation that R1 PK activity is due tocontaminant kinases, particularly CK2 (see discussion in Smith et al ., 1996). This proposal was confirmed by Langelier  et al .(1998) in experiments that convincingly demonstrate thefollowing: (1) highly purified preparations of R1 are almosttotally devoid of PK activity, (2) R1 has a very low affinity forATP and (3) the N terminus is a substrate for CK2 which canphosphorylate R1 even after SDS–PAGE and renaturation onblot. This study supports and extends Langelier’s findings bydemonstrating that trace amounts of CK2 present in R1immunoprecipitated from mammalian or bacterial extracts cangivemisleading‘autophosphorylation’resultsinthefrequentlyused immunocomplex kinase assay. These results thereforestrengthen the conclusion that the HSV R1 N terminus is nota protein kinase by confirming that all previous results in botheukaryotic and prokaryotic systems can be explained bycontaminant kinase activities, principally CK2.CK2 is a ubiquitous serine  threonine kinase present in boththe nucleus and cytoplasm of eukaryotic cells. The enzyme isa tetramer comprising two non-identical alpha and betahomodimers with monomeric molecular masses of 40 and25 kDa respectively. The alpha subunit provides the phospho-transferase function of the enzyme and the beta subunit, whichis autophosphorylated, plays a regulatory role (for review seeEdelman  et al ., 1987). CK2 has many nuclear and cytoplasmicsubstrates and mayact to regulatethe activity, stability and  orintracellular location of many cell growth control proteins. Thebeta subunit is involved in protein–protein interactions withsubstrates and has been shown to form a stable complex withtwo of these, p53 (Filhol  et al ., 1992) and DNA topoisomeraseII (Bojanowski  et al ., 1993).Initial experiments demonstrated that 1R1 was an excellentsubstrate for CK2 and the majority of 1R1 phosphorylationsites were mapped to the N-terminal domain between residues1 and 245. CK2 can phosphorylate either serines or threoninesat a variety of motifs with a broad consensus of S  TXXD  EX,although, in the majority of sites, there are three consecutiveacidic residues following the phosphate acceptor (Pearson &Kemp, 1991). Located within the N termini of both 1R1 and2R1 is a conserved block of residues (amino acids 190–237)consisting mostly of serines and aspartic acids and thisprobably provides CK2 phosphorylation sites. The highlyconserved region could be functionally important and CK2phosphorylation of serines may regulate an activity of the N-terminal domain  in vivo .Langelier  et al . (1998) proposed that R1 and CK2 mayinteract but results presented here consistently provided noevidence for a stable interaction between R1 and CK2.Strikingly,they indicated that 1R1 was readily phosphorylatedby trace amounts of CK2 contaminating the immuno-precipitates and suggest an alternative explanation for themutagenesis studies by Cooper  et al . (1995) in which fouramino acid inserts at residues 22 and 112 abrogated R1PKactivity whereas similar insertions at residues 257, 262, 292and 343 had no effect. Immunoprecipitated in112R1, thepolypeptide with a four amino acid insert at residue 112, wasa poor substrate for CK2, possibly because the insert affectedits structural integrity resulting in the inefficient display of phosphate acceptor sites. The same insertion at residue343 caused no structural alterations that affected CK2phosphorylation.Peptide phosphorylation assays identified CK2 contami-nation of 1R1 immunoprecipitated from infected cell extractsbut failed to detect any CK2 activity in R1 proteinsimmunoprecipitated from bacterial extracts. However, West-ern blotting indicated the presence of potential CK2 alpha andbeta subunits in bacterial extracts suggesting the existence of a prokaryotic CK2 homologue, although further studies will beneeded to confirm this. The peptide phosphorylation assaywas optimized for the eukaryotic enzyme and the putative  E . coli  kinase may not be fully active under these conditions; itsphosphorylation motifs and  or cation and pH requirementsmay differ. Such differences would explain the low levels of activity consistently detected with bacterially expressed R1proteins (Conner  et al ., 1992; Cooper  et al ., 1995; Langelier  etal ., 1998), since R1 would not be efficently phosphorylated by E .  coli  CK2. Theauthor is grateful to DrOdile Filhol (CNRS, Grenoble, France) forthe kind gifts of purified casein kinase 2 and casein kinase 2 antisera and BEHE  Phosphorylation of HSV R1 by CK2   Phosphorylation of HSV R1 by CK2 to Dr Howard Marsden (Institute of Virology, Glasgow) for synthesis of the peptide substrate. This study was funded in part by project grantsupport from The Wellcome Trust. References  Ali, M. A. 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(1992 b ).  Theunique N terminus of the herpes simplex virus type 1 large subunit is notrequired for ribonucleotide reductase activity.  Journal of General Virology 73 , 103–112. Conner, J., Furlong, J., Murray, J., Meighan, M., Cross, A., Marsden, H.& Clements, J. B. (1993).  Herpes simplex virus type 1 ribonucleotidereductase large subunit: regions of the protein essential for subunitinteraction and dimerisation.  Biochemistry  32 , 13673–13680. Conner, J., Marsden, H. & Clements, J. B. (1994 a ).  Ribonucleotidereductase of herpesviruses.  Reviews in Medical Virology  4 , 25–34. Conner, J., Cross, A., Murray, J. & Marsden, H. (1994 b ).  Identificationof structural domains within the large subunit of herpes simplex virusribonucleotide reductase.  Journal of General Virology  75 , 3327–3335. Conner, J., Murray, J., Cross, A., Clements, J. B. & Marsden, H. (1995). 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Edelman, A. M., Blumenthal, D. K. & Krebs, E. G. (1987).  Proteinserine  threonine kinases.  Annual Review of Biochemistry  56 , 567–613. Filhol, O., Baudier, J., Delphin, C., Loue-Mackenbach, P., Chambaz,E. M. & Cochet, C. (1992).  Casein kinase II and the tumor suppressorprotein P53 associate in a molecular complex that is negatively regulatedupon P53 phosphorylation.  Journal of Biological Chemistry  267 ,20577–20583. Heineman, T. C. & Cohen, J. I. (1994).  Deletion of the varicella-zostervirus large subunit of ribonucleotide reductase impairs growth of thevirus.  Journal of Virology  68 , 3317–3323. Jacobson, J. G., Leib, D. A., Goldstein, D. J., Bogard, C. L., Schaffer,P. A., Weller, S. K. & Coen, D. M. (1989).  A herpes simplex virusribonucleotide reductase deletion mutant is defective for productive andreactivatable latent infections of mice and for replication in mouse cells. Virology  173 , 276–283. 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(1994).  Specific inhibition of herpes virus replication by receptormediatedentryofanantiviralpeptidelinkedto Escherichiacoli enterotoxinB subunit.  Proceedings of the National Academy of Sciences ,  USA  91 ,8994–8998. Paradis, H., Gaudreau, P., Massie, B., Lamarche, N., Guilbault, C.,Gravel, S. & Langelier, Y. (1991).  Affinity purification of active subunit1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting aprotein kinase activity.  Journal of Biological Chemistry  266 , 9647–9651. Pearson, R. B. & Kemp, B. E. (1991).  Protein kinase phosphorylation BEHF
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