Specific Phosphorylation of Exogenous Protein and Peptide Substrates by the Human Cytomegalovirus UL97 Protein Kinase

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathioneS-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified32P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathione S-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified 32 P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P؉5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P؉5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.
Protein kinases regulate a multitude of biological processes and accordingly can act with exquisite specificity. Mechanisms for ensuring specificity include protein modules for binding specific substrates, regulation via interactions with specific ligands, phosphorylation and dephosphorylation, and sequence and structural determinants of the active site (1)(2)(3). A protein kinase with unusual substrate specificity is UL97. This enzyme, which is the product of the UL97 open reading frame of human cytomegalovirus (HCMV), 1 can phosphorylate nucleoside analogues such as the antiviral drugs acyclovir and ganciclovir (4 -6). UL97 is also unusual in terms of its primary sequence. It is a member of the HvU L protein kinase family, which, although sharing many motifs typical of protein kinases, is relatively divergent (7)(8)(9). Within this family, UL97 diverges even further, so that it was not immediately assumed to be a protein kinase (10). Subsequently, it was shown that UL97 can autophosphorylate on serines and threonines (11). The autophosphorylation activity has uncommon features including a rather high optimal pH (9.5), high optimal NaCl (1.5 M), and a preference for Mn 2ϩ (11). However, its exogenous protein substrates and, accordingly, its specificity for such substrates have remained unknown.
UL97 is also interesting for its role in viral infection and as an antiviral drug target. Although not absolutely essential, UL97 is critical for HCMV replication in cell culture (12). There is evidence that UL97 plays roles in viral DNA synthesis, virus assembly, and egress of assembled nucleocapsids from the nucleus (13,14). 2 A homologous kinase from another herpesvirus, herpes simplex virus, is important, at least in certain cells, for viral gene expression (15). UL97 is a target for antiviral drugs, both via its ability to activate nucleoside analogs such as ganciclovir (4 -6) and as a target for inhibition. Recently, a new antiviral drug, maribavir, has been shown to be a potent and selective inhibitor of HCMV replication in cell culture (13) and in human subjects. 3 Maribavir is a specific inhibitor of UL97 protein kinase activity (this report), 3 and indeed its anti-HCMV activity is due to its effects on UL97 2,3 . Thus, UL97 is a target for both established and promising new antiviral compounds.
To investigate the specificity of UL97 for exogenous protein and peptide substrates, we first identified an exogenous protein substrate for this enzyme, calf thymus histone H2B, and then mapped the sites of phosphorylation to specific serine residues. Peptides containing these sites were synthesized and, using a new, chimeric electrophoresis technique, were assayed as substrates for UL97. One of these peptides was phosphorylated extensively by both UL97 and cyclic AMP-dependent protein kinase (PKA) but with utilization of different serines and with susceptibility to different inhibitors. Remarkably, the extent of phosphorylation of peptides depended critically on the residue lying 5 positions downstream of the serine. Our results identify protein and peptide substrates of UL97 that could abet assays for novel inhibitors and reveal new information regarding its unusual substrate specificity, both of which could aid in the discovery of new anti-HCMV drugs.
Purification of Baculovirus-expressed GST-UL97 and GST-UL97K355Q-All column materials were purchased from Amersham Biosciences. For initial studies, baculovirus-expressed GST-UL97 and GST-UL97K355Q were purified as detailed previously (11). Subsequently, to eliminate a contaminating protein kinase activity that was active on certain substrates, the previous protocol was modified as follows. The protein that eluted from a glutathione-Sepharose column was bound to a Q-Sepharose column, previously equilibrated with elution buffer (EB; 50 mM Tris, pH 8.0, 2 mM dithiothreitol, 2 mM EDTA, 10% glycerol) with 50 mM NaCl (EB-50). The Q-Sepharose was washed with 30 column volumes of EB-50 and 30 column volumes of EB-150 (EB with 150 mM NaCl), and protein was eluted with EB-500. This material was loaded onto a phenyl-Sepharose column (equilibrated with EB-1000), and the column was washed with 30 column volumes of EB-500 and 10 volumes of EB-300 with 0.1% Triton X-100. GST-UL97 was eluted with EB-0 with 0.1% Triton X-100, concentrated with a Centricon-30 concentrator, and dialyzed for 2 h against EB-50. The resulting protein was apparently homogeneous based on SDS-PAGE. Protein concentration was determined by amino acid analysis performed by Angelo Dickerson at the Molecular Biology Core Facility of the Dana-Farber Cancer Institute, and the protein was stored in aliquots at Ϫ80°C.
Protein Kinase Assays-For phosphorylation of calf thymus histone H2B by UL97, assays were performed in 50 mM Tris-HCl (pH 9.0), 300 mM NaCl, 10 mM MgCl 2 , 2 mM dithiothreitol, and 100 M ATP), with 5 Ci/sample [␥-32 P] ATP at Ͼ5000 Ci/mmol (ICN), unless otherwise specified, and various concentrations (determined by amino acid analysis) of calf thymus histone H2B (Roche Molecular Biochemicals; "electrophoretically homogeneous") and selected amounts of GST-UL97, as specified. Synthetic peptides (200 M) were phosphorylated under the same conditions except that either 300 ng of GST-UL97 or PKA were used, MgCl 2 concentration was 20 mM, dithiothreitol concentration was 5 mM, and there was no NaCl. These buffer conditions were found to be optimal for GST-UL97 phosphorylation of peptides. 4 The reactions were incubated at 37°C for 1 h, unless otherwise specified, and terminated by the addition of 6ϫ SDS sample buffer. The samples were boiled for 3 min. Radiolabeled histones and protein kinases were separated by 15% SDS-PAGE. PAGE of peptides is described below. The resulting gels were dried, the bands were visualized by autoradiography and excised, and the radioactivity was quantified by liquid scintillation counting.
Phosphoamino Acid Analysis-Histone H2B (50 g) was phosphorylated, as described above, and separated from GST-UL97 by SDS-PAGE. The portion of the gel containing H2B was prepared for phosphoamino acid analysis as previously described (11); spotted onto a cellulose thin layer plate with a mixture of unlabeled phosphoserine, phosphothreonine, and phosphotyrosine; and subjected to electrophoresis (1200 V for 30 min) at pH 3.0 (acetic acid/pyridine/water, 75:5:920) in one dimension. Phosphoamino acid standards were visualized with 0.2% ninhydrin in acetone, and radioactivity was detected by autoradiography.
Tryptic Digestion and Extraction of Peptide Fragments-Phosphorylated 32 P-H2B (1 mg) was resolved by SDS-PAGE, and after the gel was dried, it was detected by autoradiography. The band containing phosphorylated H2B was excised and placed in a microcentrifuge tube. To reduce the concentrations of SDS and other contaminants that can inhibit trypsin, the slices were subjected to two cycles of dehydration, rehydration, and removal of liquid (17). Following a third dehydration, the slices were suspended in 100 mM ammonium bicarbonate containing 50 g TPCK-treated trypsin (sequencing grade; Roche Molecular Biochemicals). The phosphorylated protein was digested overnight at 37°C. More TPCK-treated trypsin (25 g) was added, and incubation was continued for an additional 6 h. Then excess ammonium bicarbonate was removed by evaporation under vacuum. The peptides were extracted into 400 l of acetonitrile/formic acid (1:1) by intermittently vortexing for 20 min. This step was repeated three times to maximize extraction. The extracted peptides were pooled and dried.
40% Alkaline Gel Electrophoresis-32 P-Labeled H2B tryptic peptides were loaded onto a 40% (w/v) alkaline acrylamide gel and electrophoresed overnight at 5 mA as described previously (18 -20). The resulting gel was dried, radioactivity was detected by autoradiography, radioactive bands were excised, and peptides were extracted into 0.1% trifluoroacetic acid at 4°C for 3 h with vigorous shaking. The eluant was removed and reserved. The gel slices were further extracted overnight at 4°C with fresh 0.1% trifluoroacetic acid. The two eluants were combined and dried by evaporation.
Reverse Phase-High Performance Liquid Chromatography (RP-HPLC)-Extracted peptides were dissolved in 0.1% (v/v) trifluoroacetic acid, and the solutions were adjusted to pH Ͻ2 with 10% trifluoroacetic acid. RP-HPLC was performed on a C18 column (Vydac218TP52) by James Lee at the Molecular Biology Core Facility, Dana-Farber Cancer Institute. The peptides were eluted with a linear gradient from 0 to 70% acetonitrile with 0.1% trifluoroacetic acid. Fractions were collected every 30 s, and radioactivity was monitored by Cerenkov counting.

Identification of Phosphorylation Sites by Sequencing and Mass
Spectrometry-The radioactive HPLC fractions were analyzed by peptide sequencing and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) by James Lee at the Molecular Biology Core Facility, Dana-Farber Cancer Institute. For sequencing, phosphopeptides were coupled to Immobilon-AA (Millipore Corp.) according to the manufacturer's instructions prior to sequencing. Sequencing was performed on an Applied Biosystems 477A system at the Molecular Biology Core Facility (Dana-Farber Cancer Institute) using standard techniques except that 90% (v/v) methanol containing 15 ml of 85% (w/v) phosphoric acid/100 ml was employed for residue extraction because this gave better recovery of phosphorylated derivatives. An in-line split diverted a portion of each sequencing cycle to a fraction collector to determine 32 P content by liquid scintillation counting while the remaining portion was employed for residue identification by RP-HPLC.
Peptide Synthesis-Peptides were synthesized by Dr. Charles Dahl of the Biopolymer Facility (Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School). Each peptide was verified by laser desorption mass spectrometry for mass composition, and the concentration of each solution was quantified by amino acid analysis.
Electrophoresis of Phosphorylated Peptides on Chimeric Polyacrylamide Gels-In this chimeric gel system, the stacking gel is the standard 5% stacking gel for Tris-glycine SDS-PAGE (21), whereas the resolving gel and running buffer have the same composition used for alkaline PAGE (18,20) except that 30% acrylamide (acrylamide/bisacrylamide ϭ 30:0.037) was used. When appropriate, the radioactive peptides were excised, extracted, and analyzed by mass spectrometry and amino acid sequencing, as described above.

Phosphorylation of Histone H2B by UL97 Protein Kinase-
HCMV UL97 is an unusual protein kinase that autophosphorylates on serine and threonine (11). To find exogenous substrates of UL97 and study its substrate specificity, we tested a number of common substrates of protein kinases. Of these, histones H1, H2B, and H3 and histone mixtures were most robustly phosphorylated by purified GST-UL97. To investigate the specificity of this phosphorylation further, we initially tested the phosphorylation of calf thymus histone H2B in low salt (Ͻ5 mM NaCl). Under these conditions (Fig. 1A, lane 1), H2B was efficiently radiolabeled in the presence of [␥-32 P]ATP and GST-UL97 (which also became radiolabeled due to autophosphorylation; the more faintly labeled species between GST-UL97 and H2B co-migrates with histone H1 and is prob-ably a contaminant of the commercial H2B preparation). Much less phosphorylation of either H2B or GST-UL97 was observed in the presence of maribavir (lane 2), a selective inhibitor of UL97 kinase activity 3 (see below). Similarly, much less radiolabeling was observed with a mutant form of GST-UL97 (K355Q) that contains glutamine instead of lysine at residue 355 (lane 3). Lys-355 corresponds to the invariant lysine in subdomain II of protein kinases that is crucial for enzyme activity (22), and this substitution eliminates autophosphorylation (11) (Fig. 1A, lane 3). These results indicated that most of the phosphorylation of H2B in Fig. 1A was due to GST-UL97 and not a contaminant in the enzyme preparation. However, neither maribavir, the K355Q mutation, nor both (lane 4) completely eliminated radiolabeling of H2B. This residual phosphorylation was also observed when H2B was incubated in kinase buffer in the absence of GST-UL97 (Fig. 1B, lanes 1 and 2), indicating that the commercial preparation of histone H2B used in these experiments contains small amounts of a contaminating kinase activity. When the NaCl concentration was increased to 300 mM, the residual radiolabeling in the absence of GST-UL97 was eliminated (Fig. 1B, lane 3, and Fig. 1C, lane 2), and phosphorylation by GST-UL97 was eliminated by the K355Q substitution and by maribavir (Fig. 1C, lanes 3 and 4). Under these conditions, H2B phosphorylation by GST-UL97 remained strong (Fig. 1C, lane 1), being reduced only about 20% (Fig. 1D). Higher concentrations of NaCl, up to 1 M, reduced but did not eliminate H2B phosphorylation by GST-UL97 (Fig. 1D) and stimulated autophosphorylation (11). 4 Subsequent experiments with H2B were performed at 300 mM NaCl.
Stoichiometry of H2B Phosphorylation-To determine the stoichiometry of H2B phosphorylation, a time course study was performed (Fig. 2). After incubation at 37°C for 1 h, the incorporation of phosphate reached a maximum of nearly 2 mol of phosphate/mol of H2B. The addition of more ATP or more enzyme after the reaction had reached this value did not increase the incorporation of phosphate, 4 indicating that the phosphorylation of H2B by UL97 was complete.
Analysis of Phosphorylated Sites in H2B-UL97 is known to autophosphorylate on serines and threonines (11). To identify the amino acid(s) of H2B phosphorylated by GST-UL97, 32 Plabeled H2B was hydrolyzed, and the resulting amino acid mixture was separated by thin layer electrophoresis. Essentially all the radioactive amino acids co-migrated with phosphoserine (Fig. 3A).
To identify the specific H2B residues phosphorylated, 32 Plabeled H2B that had been phosphorylated by GST-UL97 was digested with trypsin. The resulting peptides were resolved through a 40% alkaline polyacrylamide gel to separate phosphorylated species from corresponding nonphosphorylated peptides (20). As shown in Fig. 3B, there were five major species (TP1-TP5) that contained Ͼ95% of the radioactivity. Each of

FIG. 3. Phosphoamino acid and phosphopeptide analysis.
A, phosphoamino acid analysis. H2B was phosphorylated by GST-UL97, gel-purified, hydrolyzed, and subjected to thin layer electrophoresis with phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (P-Tyr) standards. The positions of the phosphoamino acid standards were visualized with ninhydrin staining (as indicated to the right), and the position of the radiolabeled peptide was visualized by autoradiography. B, tryptic phosphopeptides. H2B was phosphorylated by GST-UL97, gel-purified, and digested with TPCK-trypsin. The resulting phosphopeptides were resolved on a 40% alkaline acrylamide gel and visualized by autoradiography. The peptides were designated TP1-TP5 as indicated to the right. these five species was excised, eluted, and purified by RP-HPLC. Single peaks of radioactivity were detected for TP1-TP4, whereas three peaks of radioactivity were found for TP5 (TP5-A, TP5-B, and TP5-C). Each radioactive HPLC fraction was sequenced by Edman degradation, and a portion of the eluant from each cycle was collected and monitored for radioactivity, to identify which residue was phosphorylated (Fig. 4).
Sequence data consistent with a single phosphorylated species were obtained for all fractions except TP2, which yielded data consistent with a major species and minor species, both of which contained a serine residue at position 6. To determine which of these two species was phosphorylated or whether both were, the TP2 fraction was analyzed by MALDI-TOF. The spectrum contained a species with a mass corresponding to a  monophosphorylated form of the major species predicted from the H2B sequence (accession number 70719), but not the minor species, and one corresponding to a nonphosphorylated form of the minor species but not the major species (Table I). The MALDI-TOF spectrum of each of the other HPLC fractions contained one species with a mass corresponding to a monophosphorylated form of the sequenced peptide (Table I).
Taken together, these results identify five different serine residues as sites of phosphate incorporation: Ser-6 (TP2 and TP5-B, the latter being the complete digestion product), Ser-38 (TP1 and TP3, the latter being the complete digestion product), Ser-87 (TP4), Ser-112 (TP5-A), and Ser-124 (TP5-C). These results are consistent with the results of phosphoamino acid analysis (Fig. 3A) that showed only phosphoserine. The five sites were utilized to differing extents. Ser-38 (42% of the radioactivity), Ser-87 (24%), and Ser-6 (16%) were the three most utilized by UL97, whereas Ser-112 and Ser-124 contained only 10 and 7% of the radioactivity, respectively (Table I). The relatively weak phosphorylation at certain sites accounts for the discrepancy between the 2 mol of phosphate incorporated/ mol of H2B and the five phosphorylation sites detected. Interestingly, the Ser-38, Ser-87, and Ser-6 sites contain diverse residues at all positions around the phosphorylated serine except at the position 5 residues downstream (Pϩ5), where all three peptides contain a basic residue (Lys or Arg).
Phosphorylation of Synthetic Peptides-Peptides corresponding to portions of H2B containing each of the five phosphorylated serines (SP-6, SP-38, SP-87, SP-112, and SP-124) were synthesized and tested as substrates for UL97. The standard protocol to monitor peptide phosphorylation involves a filterbinding assay (23). However, such an assay was problematic, because autophosphorylation of UL97 obscured the signal due to peptide phosphorylation. 4 To overcome this problem, a new "chimeric" gel electrophoresis technique was developed, in which an SDS-containing "stacking" gel from standard Trisglycine SDS-PAGE was combined with a detergent-free resolving gel from an alkaline acrylamide gel (18,20). Using this system to separate enzyme from peptide, we found that a synthetic peptide containing Ser-38 (SP38) was readily phosphorylated by GST-UL97 (Fig. 5). SP-87, containing Ser-87, was also phosphorylated, but not as extensively. Faint phosphorylation of peptide SP-6 could also be discerned and, on longer exposure, weak phosphorylation of the other two peptides. This correlates well with the data on phosphorylation of full-length H2B, where Ser-38 and Ser-87 were the most highly phosphorylated sites, followed by SP6.
Comparison of Specificities of PKA and UL97-Phosphorylation of H2B by PKA has been extensively studied (24 -26). PKA phosphorylates both Ser-32 and Ser-36 of H2B (26), the latter of which is contained in peptide SP38. To compare the specificities of PKA and UL97, the phosphorylation of SP-38 (RKESYSVYVYKV) by GST-UL97 and PKA was studied (Fig.  6A). Phosphorylation of SP-38 by UL97, but not PKA, was sensitive to maribavir, whereas phosphorylation by PKA, but  1 and 2) or 187 (lanes 3 and 4) or residue 3 of UL97 5 (lanes 5 and 6) were synthesized, phosphorylated by UL97, and resolved on chimeric polyacrylamide gels, and phosphorylated species were visualized by autoradiography. In each panel, the upper phosphorylated band present in both lanes is phosphorylated GST-UL97, and the lower band (visible only in the odd-numbered lanes that contain wild type peptides) is the phosphorylated peptide. not UL97, was sensitive to PKI, a specific pseudosubstrate inhibitor of PKA (27,28). Moreover, phosphorylation of SP-38 by UL97 was sensitive to the K355Q substitution. When the phosphorylated peptides were analyzed by MALDI-TOF, only monophosphorylated forms were observed. 4 Sequence analysis revealed that PKA phosphorylated the peptide specifically on Ser-36 and UL97 phosphorylated the peptide specifically on Ser-38, as predicted (Fig. 6B). Thus, presented with the same peptide, UL97 and PKA each have strong specificity for a particular serine as well as distinct inhibitor specificities.
Importance of the Pϩ5 Position-As noted above, the three sites in H2B and the two H2B-derived peptides most extensively phosphorylated by UL97 contain a Lys or Arg in the Pϩ5 position. Interestingly, the site on UL97 that is most extensively autophosphorylated by UL97 5 also contains a basic residue at Pϩ5 (Fig. 5B). The results raised the possibility that the rather distal Pϩ5 position might be important for phosphorylation by UL97. To test this hypothesis, synthetic peptides derived from the two most extensively phosphorylated sites in H2B and the most extensively autophosphorylated site in UL97, 5 but containing an Ala in place of Arg or Lys at the Pϩ5 position, were synthesized. Phosphorylation of these mutant peptides was essentially eliminated, compared with their wild type counterparts (Fig. 7). Thus, phosphorylation of three different peptides by UL97 critically depends on the identity of the residue at the Pϩ5 position. DISCUSSION The work described here documents the phosphorylation by purified HCMV UL97 of an exogenous substrate, histone H2B, and of H2B-derived peptides on specific Ser residues. To show conclusively that phosphorylation was performed by UL97 rather than by a contaminant in the enzyme or H2B preparation, we not only purified the enzyme extensively but also used both a catalytically inactive, mutant form of UL97 (K355Q) and a specific UL97 inhibitor, maribavir. HCMV UL97 is a member of the HvU L protein kinase family (7,10). It has been reported that certain members of this family are capable of phosphorylating various exogenous substrates in vitro, including certain preparations of histones (8, 29 -33). However, in these reports, the enzymes were not purified extensively and/or it was not shown that an inactivating point mutation or a specific inhibitor eliminated phosphorylation of the exogenous substrates. Indeed, in two cases (32,33), mutant forms akin to UL97 K355Q retained substantial kinase activity, suggesting that the enzyme preparations were contaminated with other kinases. Regardless, to our knowledge, the work presented here is the first to demonstrate phosphorylation of an exogenous substrate by UL97, an especially divergent member of this already divergent family of protein kinases, and the first to describe sites of phosphorylation by any member of this family.
Aside from H2B, UL97 can phosphorylate histones H3 and H1 in vitro. 4 It is not inconceivable that histones could be physiologically relevant substrates of UL97. UL97 is localized to the nucleus in HCMV-infected cells (34,35). HCMV infection is known to affect host gene expression (reviewed in Ref. 36), and other herpesviruses are known to affect chromatin condensation (reviewed in Ref. 37), both processes that can be regulated by histone phosphorylation (38). These possibilities can be tested with the aid of maribavir and UL97 deletion mutants (12).
A goal of this study was to find clues to the specificity of phosphorylation by UL97, both to understand the mechanism of specificity and to help predict intracellular substrates of this enzyme. Sites of phosphorylation of HvU L protein kinases have not previously been identified. We anticipated that peptide and protein substrates of UL97 might have unusual characteristics based on its ability to phosphorylate nucleoside analogues, its sequence divergence from protein kinases, and its high pH and NaCl optima. That the specificity of UL97 differs from that of a prototypic protein kinase, PKA, is clearly illustrated by the phosphorylation of different Ser residues when both kinases are presented with the same peptide (SP-38). The phosphorylation of the Ser corresponding to Ser-36 by PKA is consistent with the established consensus substrate for PKA, (R/K)XXS (39 -41).
More generally, for diverse protein kinases, specific residues from P-3 through Pϩ3 are most important for substrate specificity, with the PϪ3 (e.g. PKA) and Pϩ1 positions usually being the most crucial; it is highly unusual for the Pϩ5 position to be important (42). Our results reveal, however, that the sites that are most extensively phosphorylated by UL97 contain an Arg or Lys at the Pϩ5 position. Indeed, substitution of a Pϩ5 Arg or Lys with Ala essentially eliminates phosphorylation of synthetic peptides. Thus, this rather distal residue is very important for UL97. In contrast, there were no obvious similarities in other positions of extensively phosphorylated peptides (Fig. 5B). The unusual importance of the Pϩ5 position is consistent with the lack of conservation of UL97 with other protein kinases in subdomains typically involved in substrate recognition. In particular, UL97 lacks acidic residues in subdomains V and VI that are thought to stabilize binding of basic PϪ3 residues, and it does not have a readily recognizable subdomain VIII, which ordinarily makes up much of the Pϩ1 binding site of other kinases (10,22,42). At present, little is known about residues in any protein kinase that interact with the Pϩ5 position. Further studies of the substrate specificity of UL97 should advance our understanding of this unusual kinase-substrate interaction.
Our results may aid the discovery of new antiviral drugs that target UL97. The identification of exogenous substrates for UL97 permits development of assays for new inhibitors of UL97. It is especially advantageous to have identified a peptide substrate of UL97, since this can facilitate the development of high throughput assays. Moreover, peptide substrates can be the starting points for the design of peptidomimetic inhibitors of protein kinases. Such substrates can be abetted by combinatorial approaches to optimize affinity and conversion of serines to nonphosphorylatable moieties (43), followed by conversion of peptides to peptidomimetics, as has occurred in the development of protease inhibitor drugs (44). The peptide substrate, SP-38, may serve as a starting point for a novel UL97 inhibitor.