The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro.
The human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. In the presence of [gamma32P]ATP, phosphorylation of Nef occurred predominantly on serine residues. Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation. This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses. In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components.
Coates
K
K
MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, UK.
Harris
M
M
D000818:Animals; D002460:Cell Line; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C