The transmembrane domain of the large subunit of HSV-2 ribonucleotide reductase (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 reductase (ICP10) is a chimera consisting of a Ser/Thr protein kinase (PK) with 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 transformation we established cell lines that constitutively express ICP10 (JHLa1) or its TM deleted mutant p139TM (JHL15). ICP10 was associated with purified JHLa1 plasma membranes. Membrane immunofluorescence and FACS analysis with antibodies to synthetic peptides located upstream and downstream of the TM indicated that ICP10 is a membrane-spanning protein. p139TM was not associated with JHL15 plasma membranes. ICP10 kinase activity was detected in JHLa1 but not JHL15 cells as determined by immunocomplex kinase assays and metabolic labeling. JHLa1 cells displayed anchorage-independent growth whereas JHL15 cells and JHL9 cells that express a mutant deleted in the PK catalytic domain were negative. ras-GTPase activating protein (ras-GAP) was phosphorylated in JHLa1 but not JHL15 cells and GTPase activity was lower in JHLa1 than JHL15 cells. Furthermore, ICP10 but not p139TM bound the guanine nucleotide releasing factor son of sevenless 1 (Sos1) and ras-GTP (activated ras) was higher in JHLa1 than JHL15 cells. The data suggest that ICP10 constitutively increases ras activity, and its TM segment plays a critical role in transformation-related signaling pathways.
Smith
C C
CC
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
Luo
J H
JH
Hunter
J C
JC
Ordonez
J V
JV
Aurelian
L
L
D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005786:Gene Expression Regulation; D011905:Genes, ras / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000032:analysis; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics* / Q000378:metabolism; D018258:Herpesvirus 2, Human / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics; D020837:SOS1 Protein; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020703:ras GTPase-Activating Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D020837:SOS1 Protein; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D012264:Ribonucleotide Reductases; D017346:Protein Serine-Threonine Kinases