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Volume 271, Number 29, Issue of July 19, 1996 pp. 17021-17027
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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ATP and SH3 Binding Sites in the Protein Kinase of the Large Subunit of Herpes Simplex Virus Type 2 of Ribonucleotide Reductase (ICP10)

(Received for publication, December 29, 1995, and in revised form, April 24, 1996)

John W. Nelson ^§ , Jia Zhu ^¶ , Cynthia C. Smith ^¶ , Michael Kulka ^¶ and Laure Aurelian ^§¶

From the  Virology/Immunology Laboratories, the ^¶ Department of Pharmacology and Experimental Therapeutics, and the ^§ Department of Microbiology, and Molecular Cell Biology Program, University of Maryland School of Medicine, Baltimore, Maryland 21201 and the  Department of Biochemistry, The Johns Hopkins Medical Institutions, Baltimore, Maryland, 21205

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein. It consists of a ribonucleotide reductase and a serine/threonine protein kinase (PK) domain, which has three proline-rich motifs consistent with SH3-binding sites at positions 140, 149, and 396. We used site-directed mutagenesis to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys¹⁷⁶ or Lys²⁵⁹ reduced PK activity (5-8-fold) and binding of the ¹⁴C-labeled ATP analog -fluorosulfonylbenzoyl 5-adenosine (FSBA) but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu²⁰⁹ (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg²⁺ or Mn²⁺ ions, suggesting that Glu²⁰⁹ functions in ion-dependent PK activity. ICP10 bound the adaptor protein Grb2 in vitro. Mutation of the ICP10 proline-rich motifs at positions 396 and 149 reduced Grb2 binding 20- and 2-fold, respectively. Binding was abrogated by mutation of both motifs. Grb2 binding to wild type ICP10 was competed by a peptide for the Grb2 C-terminal SH3 motif, indicating that it involves the Grb2 C-terminal SH3.

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