Steady-state plasma membrane expression of human cytomegalovirus gB is determined by the phosphorylation state of Ser900.
Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser900. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser900, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser900 was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser900 trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to the trans-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.
Fish
K N
KN
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Soderberg-Naucler
C
C
Nelson
J A
JA
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003587:Cytomegalovirus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011714:Pyrans; D011817:Rabbits; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013141:Spiro Compounds; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D011714:Pyrans; D013141:Spiro Compounds; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus; C053079:tautomycin; D001224:Aspartic Acid; D012694:Serine; D010749:Phosphoprotein Phosphatases; D000409:Alanine