Induction of phosphorylation of human immunodeficiency virus type 1 Nef and enhancement of CD4 downregulation by phorbol myristate acetate.
The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) encodes a 27 to 34 kDa myristoylated protein that induces downregulation of CD4 from the cell surface and enhances virus infectivity. As shown by experiments on SIV-infected adult macaques, Nef is important in pathogenesis and disease progression. In vitro, protein kinase C (PKC) phosphorylates Nef, but the role of phosphorylation in the function and expression of this protein has not yet been determined. Here we show that in HIV type 1-infected cells, phosphorylation of Nef increased 8- to 12-fold after treatment with phorbol myristate acetate and phytohemagglutinin (PMA/PHA). Basal and PMA/PHA-induced phosphorylation occurred on serine residues of Nef and was independent of other HIV proteins. The PMA/PHA-induced phosphorylation of Nef was inhibited by bisindolylmaleimide I, a potent and specific inhibitor of PKC, but was unaffected by H89, an inhibitor of protein kinase A. In contrast, treatment with bisindolylmaleimide I did not affect the basal level of Nef phosphorylation, suggesting two different phosphorylation pathways. A PMA-insensitive CD4 mutant in which three serine residues in the cytoplasmic domain have been replaced by alanines was used to determine whether PMA-induced phosphorylation affects Nef-induced CD4 downregulation. In Nef-expressing cells, treatment with PMA enhanced downregulation of the CD4 serine triple mutant from the cell surface, suggesting that phosphorylation is important for Nef function.
Luo
T
T
Department of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Downing
J R
JR
Garcia
J V
JV
D015704:CD4 Antigens / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D010766:Phosphorylation; D010835:Phytohemagglutinins / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D054311:nef Gene Products, Human Immunodeficiency Virus
D015704:CD4 Antigens; D004791:Enzyme Inhibitors; D015693:Gene Products, nef; D007211:Indoles; D007546:Isoquinolines; D008301:Maleimides; D010835:Phytohemagglutinins; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D054311:nef Gene Products, Human Immunodeficiency Virus; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate