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Journal of Virology, December 2002, p. 11943-11952, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.11943-11952.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Relationship between Autophosphorylation and Phosphorylation of Exogenous Substrates by the Human Cytomegalovirus UL97 Protein Kinase

Moon-Chang Baek, Paula M. Krosky, and Donald M. Coen^*

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Received 30 May 2002/ Accepted 14 August 2002

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 ³²P-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.

Present address: Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.

Present address: Screening Technologies Branch, Developmental Therapeutics Program, NCI Frederick, Frederick, MD 21702.

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