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).

J.H Luo,L Aurelian

doi:10.1016/s0021-9258(19)50139-9

Abstract

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).

Highlights

Thelargesubunitofherpessimplex virus type 2 truncated protein ( ~ ~ 2 9 ~p“ro’v)ides a compelling argument ribonucleotide reductase (ICPlOi)s a chimera consist- that the PKactivity is an intrinsic property of ICPlO (Luo et ing, at the amino terminus, of a Ser/Thr prkoitneainse al., 1991)
In pJHL4 the invariant Lys in motif I1 ( L Y s ' ~w~a)s substituted by a Leu residue, thereby modifying the net charge at the site and presumablyinterferingwith phosphotransferreactions
Protein expression was verified by Monoclonal antibodies (mAbs) 30 immunoprecipitationof [35S]methionine-labeledextracts of transfected cells, and immunoprecipitates of unlabeled duplicates of the cell extracts were assayed for PK activity as described (Chunget al., 1989, 1990)

Summary

Introduction

Thelargesubunitofherpessimplex virus type 2 truncated protein ( ~ ~ 2 9 ~p“ro’v)ides a compelling argument ribonucleotide reductase (ICPlOi)s a chimera consist- that the PKactivity is an intrinsic property of ICPlO (Luo et ing, at the amino terminus, of a Ser/Thr prkoitneainse al., 1991). Fate-polyacrylamide gel electrophoresis and transfer Comparison of the predicted amino acid sequence of ICPlO to membranefilters Both ICPlO and the invariant Lyalsigned as described by Hanks et al (1988),with those of the mutant bound “C-labeled p-fluorosulfonylbenzoyl 5‘- catalytic subunitsof other PKsrevealed the presence of eight adenosine, an ATP affinity analog. Expression of thePK minigene in abacterial expression system that permitssynthesis of an enzymatically active to define the functional significance of catalytic motifs I and I1 and of the putative TM segment. They indicate that catalytic motifs I and I1 are not essential for the kinase activity of ICPlO while kinase activity is lost by deletion of sequences in the TMsegment.

Methods

Results

Conclusion