Relationship between autophosphorylation and phosphorylation of exogenous substrates by the human cytomegalovirus UL97 protein kinase

Abstract

Human cytomegalovirus encodes an unusual protein kinase, UL97, which is a member of the HvU(L) family of protein kinases encoded by diverse herpesviruses. UL97 is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. It has previously been concluded that phosphorylation of UL97 is essential for its phosphorylation of ganciclovir. We examined the relationship between autophosphorylation of UL97 and its activity on exogenous substrates. Glutathione S-transferase-UL97 fusion protein purified from insect cells was found to be already partially phosphorylated, but neither extensive autophosphorylation nor phosphatase treatment meaningfully altered the time course of its phosphorylation of the exogenous substrate, histone H2B. Sequencing and mass spectrometric analyses of (32)P-labeled tryptic peptides of the UL97 fusion protein identified nine sites of autophosphorylation, all within the first 200 residues of the protein, outside of conserved protein kinase subdomains. A peptide corresponding to the N-terminal UL97 segment that was most extensively autophosphorylated was readily phosphorylated by UL97, confirming that fusion protein sequences are not required for phosphorylation at this site. Deletion mutants lacking at least the first 239 residues exhibited drastically reduced autophosphorylation (<5%) but retained near-wild-type H2B phosphorylation activity. Baculoviruses expressing these mutants efficiently directed the phosphorylation of ganciclovir in insect cells. Taken together, these results identify the autophosphorylation sites of a herpesvirus protein kinase and show that autophosphorylation of UL97 is not required for phosphorylation of exogenous substrates.

FIG. 1.

Electrophoretic behavior and phosphorylation state of UL97. GST-UL97 purified from insect cells expressing either wt GST-UL97 (lanes 1 and 2) or mutant GST-UL97K355Q (lane 3) was analyzed by SDS-PAGE either after treatment with (lane 2) or without (lanes 1 and 3) lambda phosphatase.

FIG. 2.

Stoichiometry of phosphorylation of GST-UL97. A total of 400 ng of either untreated GST-UL97 (○) or GST-UL97 that had been treated with lambda phosphatase (•) were incubated in kinase buffer with radiolabeled ATP. Aliquots were removed at the indicated times, and ³²P incorporation was measured.

FIG. 3.

Effect of lambda phosphatase treatment on rates of autophosphorylation and H2B phosphorylation. (Top) Flow chart of the experiment. (Bottom) Results of the experiment. A total of 800 ng of GST-UL97 was dephosphorylated by lambda (λ) phosphatase and, as described in Materials and Methods, half was incubated with unlabeled (“cold”) ATP for 30 min (PPase/ATP; ▵) and the other half was incubated in kinase buffer without ATP (PPase; ▴). Then, 5 μg of histone H2B and radiolabeled (“hot”) ATP were added to each preparation, with additional unlabeled (“cold”) ATP added to the PPase preparation. Aliquots were removed at the indicated times and the amount of phosphate incorporated per mol of GST-UL97 (bottom, left panel; Auto) or histone H2B (bottom, right panel; H2B) were measured.

FIG. 4.

Tryptic phosphopeptides of GST-UL97. Phosphatase-treated GST-UL97 (A) and untreated GST-UL97 (B) were autophosphorylated with radiolabeled ATP and digested with trypsin. The resulting peptides were resolved on a 30% (wt/vol) alkaline acrylamide gel. After electrophoresis, the gel was dried onto filter paper and autoradiographed. The positions of the six major species (UL97-1 to UL97-6) are indicated immediately to the right of the autoradiogram. To the right of that are lines and numbers indicating the residues of UL97 that are represented in the various autophosphorylated peptides and the number of phosphate groups (P) in each peptide. Negative numbers refer to residues upstream of UL97 in the GST moiety. Below is a schematic representation of GST-UL97 showing the relative locations of the N-terminal and kinase domains.

FIG. 5.

Sequence analysis of UL97 phosphopeptides. Tryptic phosphopeptides UL97-1 to UL97-6 (as indicated at the top of each panel) were further purified by RP-HPLC, and peak fractions were subjected to automated amino-terminal microsequencing. The material released at each cycle was split to identify the amino acid by RP-HPLC (shown at the bottom of each panel) and the radioactivity by scintillation counting (indicated by black bars). Phosphorylated residues are underlined.

FIG. 6.

Phosphorylation of SP-1 by GST-UL97. Synthetic peptide SP-1 (200 μM) was phosphorylated in kinase buffer for 30 min by using 200 ng of GST-UL97 (lanes 1 and 3) or GST-UL97K355Q (lane 2), either in the presence (lane 3) or absence of 3 μM maribavir (lanes 1 and 2). The reaction products were resolved by electrophoresis on a chimeric polyacrylamide gel, which was analyzed by a phosphorimager. The locations of GST-UL97 and phosphopeptide are indicated to the left.

FIG. 7.

Sequence analysis of phosphorylated SP-1. After phosphorylation of SP-1 by GST-UL97, the radiolabeled peptide was resolved from GST-UL97 by gel electrophoresis, extracted from the gel, and analyzed by amino acid sequencing as described in the legend to Fig. 5, with the radioactivity at cycle 3 set to 100%.

FIG. 8.

Differential effects of N-terminal truncations on autophosphorylation and H2B phosphorylation. Equivalent amounts of wt GST-UL97 or N-terminal truncation mutants, ΔN239 and ΔN277, were incubated with histone H2B and radiolabeled ATP in kinase buffer. At various times, aliquots were analyzed for autophosphorylation or H2B phosphorylation. (A) Phosphorimage of an SDS-polyacrylamide gel of radiolabeled products after 30 min of incubation. The GST-UL97 proteins are indicated at the top of the panel, and the positions of autophosphorylated GST-UL97 (Auto-P) and phosphorylated H2B (H2B-P) are indicated to the left. (B) Time course of the reactions for wt GST-UL97 (WT; open symbols) and ΔN239 (solid symbols) for autophosphorylation (Auto-P; triangles) and H2B phosphorylation (H2B-P; diamonds). The maximal incorporation for autophosphorylation and H2B phosphorylation by wt enzyme was set as 100%.

FIG. 9.

Large N-terminal truncations of UL97 do not decrease GCV anabolism. Insect cells were mock infected or infected with recombinant baculoviruses expressing the indicated GST-UL97 proteins. (A) Lysates were prepared at 48 h p.i., and proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was reacted with an anti-GST antibody, which was detected with horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin antibody. The versions of GST-UL97 expressed in each lysate are indicated at the top of the panel. (B) At 24 h p.i., cells were pulsed with radiolabeled GCV for the times indicated, lysates were prepared, and radiolabled GCV anabolites were measured by using a filter-binding assay. WT, wild type; Mock, mock infected.