Intracellular internalization and signaling pathways triggered by the large subunit of HSV-2 ribonucleotide reductase (ICP10).
The large subunit of the HSV-2 ribonucleotide reductase (RR) (ICP10) is a chimera consisting of a serine threonine (Ser/Thr) protein kinase domain at the amino terminus and the RR domain at the carboxy terminus. Transformed human cells that constitutively express ICP10 (JHLa1) were stained with anti-LA-1 antibody (recognizes ICP10 amino acids 13-26) and immunogold-conjugated goat anti-rabbit IgG and were examined by electron microscopy. ICP10-associated gold particles were observed on the cell surface and in structures with ultrastructural characteristics of endocytic vesicles, multivesicular bodies, and lysosomes, consistent with endocytic internalization. ICP10 was also associated with the cytoskeleton fraction of JHLa1 cells and, at least in part, it colocalized with actin filaments. This was evidenced by immunoprecipitation of [35S]methionine-labeled cell fractions and immunofluorescent staining of Triton-treated cells with anti-LA-1 antibody and phalloidin. Endocytic localization of gold particles was not seen in cells that constitutively express the ICP10 transmembrane (TM)-deleted mutant p139TM (JHL15). p139TM did not associate with the cytoskeleton and was almost entirely localized within the cytoplasm. raf and Erk evidenced decreased mobility consistent with an activated state in JHLa1, but not JHL15, cells, and chloramphenicol acetyl transferase (CAT) expression from a c-fos/cat hybrid construct was significantly increased in JHLa1 but not JHL15 cells. The data indicate that effector molecules downstream of ras are activated in JHLa1 cells and the ICP10 TM segment plays a critical role in ICP10 intracellular localization and its ability to activate signaling pathways. This behavior is analogous to that of an activated growth factor receptor kinase.
Hunter
J C
JC
Virology/Immunology Laboratories, University of Maryland School of Medicine, Baltimore 21201, USA.
Smith
C C
CC
Bose
D
D
Kulka
M
M
Broderick
R
R
Aurelian
L
L
D000199:Actins / Q000032:analysis; D002458:Cell Fractionation; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry; D011992:Endosomes / Q000737:chemistry / Q000648:ultrastructure; D016762:Genes, fos / Q000235:genetics; D018258:Herpesvirus 2, Human / Q000201:enzymology*; D006801:Humans; D008247:Lysosomes / Q000737:chemistry / Q000648:ultrastructure; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019908:Proto-Oncogene Proteins c-raf; D012264:Ribonucleotide Reductases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation
D000199:Actins; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D012264:Ribonucleotide Reductases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases