ATP and SH3 binding sites in the protein kinase of the large subunit of herpes simplex virus type 2 of ribonucleotide reductase (ICP10).
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 rho-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.
Nelson
J W
JW
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Zhu
J
J
Smith
C C
CC
Kulka
M
M
Aurelian
L
L
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D018698:Glutamic Acid; D006801:Humans; D007668:Kidney; D008239:Lysine; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011392:Proline; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology*; D014709:Vero Cells; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008345:Manganese; D000255:Adenosine Triphosphate; D011392:Proline; C100671:ICP10 protein, herpes simplex virus type 2; D012264:Ribonucleotide Reductases; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium; D008239:Lysine