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 E scherichia coil as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinas es in vitro was examined by incubation of purified GST-Nef fusion prot eins, immobilized on glutathione-agarose beads, with cytoplasmic extra cts from a number of human cell lines. In the presence of [gamma(32)P] 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 furthe r by the demonstration that purified PKC was also able to phosphorylat e Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-term inal GST or 6-histidine tag in Spodoptera frugiperda insect cells by r ecombinant baculo-viruses. In extracts from Jurkat T cells and U937 mo nocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-a ssociated protein was inhibited by heparin and is thus likely to be me diated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in reg ulating both Nef function and the physical interactions between Nef an d cellular components.