The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).
The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a chimera consisting, at the amino terminus, of a Ser/Thr protein kinase (PK) with features of a signal peptide and a transmembrane (TM) helical segment, and at the carboxy-terminus, of the ribonucleotide reductase (Chung et al., 1989, 1990). Membrane immunofluorescence of ICP10 transformed cells with antibodies to synthetic peptides located upstream or downstream of the TM indicates that ICP10 is a membrane-spanning protein. Site-directed and deletion mutants were used to further characterize ICP10-PK. Mutation of Gly106 in catalytic motif I or of the invariant Lys in catalytic motif II, and deletion of both motifs (amino acids 106-178) did not eliminate kinase activity. PK activity was retained by the invariant Lys mutant expressed in bacteria and following protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to membrane filters. Both ICP10 and the invariant Lys mutant bound 14C-labeled rho-fluorosulfonylbenzoyl 5'-adenosine, an ATP affinity analog. The deletion mutant had 4-fold lower kinase activity than ICP10-PK, and it was insensitive to Mn2+, suggesting that these motifs are involved in Mn2+ activation of kinase activity. PK activity was lost by deletion of the TM segment (amino acid residues 85-106).
Luo
J H
JH
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
Aurelian
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics
D004279:DNA, Viral; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases; D011494:Protein Kinases