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J. Biol. Chem., Vol. 277, Issue 33, 29593-29599, August 16, 2002
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From the Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
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
32P-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.
Specific Phosphorylation of Exogenous Protein and Peptide
Substrates by the Human Cytomegalovirus UL97 Protein Kinase
IMPORTANCE OF THE P+5 POSITION*
,
*
This work was supported in part by National Institutes of
Health Grant RO1 AI26077 (to D. M. C.).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.
Supported in part by the Korean Science and Engineering
Foundation. Present address: Div. of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
§
Supported in part by NIH Grant T32 AI07245. Present address:
Screening Technologies Branch, Developmental Therapeutics Program, NCI-Frederick, Frederick, MD 21702.
¶
Present address: Gotham Networks, Acton, MA 01720.
To whom correspondence should be addressed: Dept. of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1691; Fax: 617-432-3833; E-mail: Don_Coen@hms.harvard.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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