Characterization of the intrinsic protein kinase activity and the kinase catalytic motifs of the large subunit of the herpes simplex virus type 2 ribonucleotide reductase

Abstract

The large subunit of Herpes Simplex Virus type 2 (HSV-2) ribonucleotide reductase (ICP10) is a chimera protein consisting of a serine/threonine protein kinase (PK) domain at the amino-terminus and a ribonucleotide reductase (RR) domain at the carboxyl-terminus. Like growth factor receptor PKs, ICP10 is myristylated, it has features of a signal peptide and putative transmembrane (TM) segment, and its PK activity is modulated by basic proteins and by antibodies to amino acid residues upstream of the TM. To further characterize this PK domain, we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29 KDa protein (designated pp29{dollar}\sp{lcub}\rm la1{rcub}){dollar} that represents a truncated portion of the ICP10 PK domain as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10 positive eukaryotic cells, and peptide mapping. pp29{dollar}\sp{lcub}\rm la1{rcub}{dollar} has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn{dollar}\sp{lcub}2+{rcub}{dollar} but not by Mg{dollar}\sp{lcub}\rm 2+{rcub}{dollar} ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine. To determine the significance of ICP10 PK catalytic motifs, site-directed and deletion mutants in PK motifs I and II, the putative signal peptide and the TM segment were used to determine the role of these elements in ICP10-PK activity. PK activity was lost by deletion of the putative TM segment (amino acid residues 85-106). However, mutation of the central Gly in PK catalytic motif I (Gly{dollar}\sp{lcub}106{rcub}{dollar}) or of the invariant Lys in PK catalytic motif II (Lys{dollar}\sp{lcub}176{rcub}{dollar}) or deletion of both of these catalytic motifs (amino acid residues 106-178) did not abolish the kinase activity as determined both in auto- and transphosphorylation assays. PK activity of the mutant deleted in domains I and II was 4-fold lower than that of the wild type ICP10 and it was insensitive to Mn{dollar}\sp{lcub}2+{rcub}{dollar}, suggesting that these motifs are involved in Mn{dollar}\sp{lcub}2+{rcub}{dollar} activation of kinase activity. The result of immunoblotting demonstrated that ICP10 complexes with GTPase activating protein (GAP). Ras GTPase activity is significantly inhibited in ICP10 transformed (JHLa1) cells. These results suggested that ICP10 may constitutively activate ras activity by blocking its down-regulation process, implying a potential signal transduction mechanism for ICP10 induced transformation.