THE PROTEIN-KINASE ACTIVITY OF THE LARGE SUBUNIT OF HERPES-SIMPLEX VIRUS TYPE-2 RIBONUCLEOTIDE REDUCTASE (ICP10) FUSED TO THE EXTRACELLULARDOMAIN OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR IS LIGAND-INDUCIBLE

The gene coding for the large subunit of herpes simplex Virus type 2 r ibonucleotide reductase (RR) (ICP10) has a unique 5' terminal domain t he product of which has a serine/threonine (Ser/Thr) protein kinase (P K) catalytic domain preceded by a transmembrane (TM) segment. Because ICP10 localizes on the cell surface and is internalized by the endocyt ic pathway like an activated growth factor receptor (Hunter et al., 19 95, Virology 210, 345-360), we asked whether it is ligand-inducible in order to examine whether it has intrinsic transphosphorylating activi ty. We constructed a chimeric expression vector that contains the extr acellular and TM domains of the epidermal growth factor receptor (EGFR ) joined to the intracellular PK and RR domains of ICP10 (pCH5) and es tablished constitutively expressing cell lines in NIH3T3 2.2 cells tha t do not express EGFR. The chimeric protein, designated p210(CH5), loc alized to the surface of these cells as determined by immunofluorescen t staining with MAb EGFR, and it bound I-125-EGF. p210(CH5) coprecipit ated with protein species p170, p120, p88, p60, p44, p34, and p25. EGF treatment activated the PK activity of p210(CH5), resulting in its au tophosphorylation and the phosphorylation of the p120, p88, and p34 sp ecies. Immunoprecipitation/immunoblotting with anti-ras-GAP antibody a nd phosphoamino acid analysis indicated that p120 is ras-GAP and it is phosphorylated on Ser/Thr residues. The identities of the phosphoryla ted p88 and p34 are still unknown. The data indicate that when fused t o a ligand-regulated extracellular domain (EGFR), the ICP10 PK auto- a nd transphosphorylating activities are ligand-inducible. These finding s support the interpretation that the ICP10 PK activity is intrinsic a nd indicate that ras-GAP is one of its phosphorylation substrates. (C) 1995 Academic Press, Inc.