1887

Abstract

The human immunodeficiency virus type 1 Nef protein was expressed in as a C-terminal fusion with glutathione -transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases 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 [γP]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 when Nef was expressed with a C-terminal GST or 6-histidine tag in 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 . 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 raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components.

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1995-04-01
2022-05-28
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