THE TRANSMEMBRANE DOMAIN OF THE LARGE SUBUNIT OF HSV-2 RIBONUCLEOTIDEREDUCTASE (ICP10) IS REQUIRED FOR PROTEIN-KINASE ACTIVITY AND TRANSFORMATION-RELATED SIGNALING PATHWAYS THAT RESULT IN RAS ACTIVATION

The large subunit of Herpes simplex virus type 2 ribonucleotide reduct ase (ICP10) is a chimera consisting of a Ser/Thr protein kinase (PK) w ith features of a transmembrane (TM) helical segment localized at the amino terminus, and the RR1 domain localized at the carboxy terminus. To elucidate the role of the TM segment in ICP10-mediated transformati on we established cell lines that constitutively express ICP10 (JHLa1) or its TM deleted mutant p139TM (JHL15). ICP10 was associated with pu rified JHLa1 plasma membranes. Membrane immunofluorescence and FACS an alysis with antibodies to synthetic peptides located upstream and down stream of the TM indicated that ICP10 is a membrane-spanning protein. p139TM was not associated with JHL(15) plasma membranes. ICP10 kinase activity was detected in JHLa1 but not JHL15 cells as determined by im munocomplex kinase assays and metabolic labeling. JHLa1 cells displaye d anchorage-independent growth whereas JHL15 cells and JHL9 cells that express a mutant deleted in the PK catalytic domain were negative. ra s-GTPase activating protein (ras-GAP) was phosphorylated in JHLa1 but not JHL15 cells and GTPase activity was lower in JHLa1 than JHL15 cell s. Furthermore, ICP10 but not p139TM bound the guanine nucleotide rele asing factor son of sevenless 1 (Sos1) and ras-GTP (activated ras) was higher in JHLa1 than JHL15 cells. The data suggest that ICP10 constit utively increases res activity, and its TM segment plays a critical ro le in transformation-related signaling pathways. (C) 1994 Academic Pre ss, Inc.