IN-VITRO BINDING AND PHOSPHORYLATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF PROTEIN BY SERINE THREONINE PROTEIN-KINASE

Although the human immunodeficiency virus type 1 (HIV-1) nef gene stil l has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to i dentify cellular proteins capable of binding to Nef, the HIV-1(LAI) ne f gene product was expressed in the bacterial vector pGEX-2T as a glut athione S-transferase (GST)-Nef fusion protein. Deletion mutants corre sponding to 86 and 35 N-terminal residues of the Nef protein were prep ared. The GST-Nef constructs were used to identify cellular kinases ca pable of interacting with Nef After incubation with a Jurkat cell lysa te, the GST-Nef constructs immobilized on glutathione-agarose beads bo und to cellular kinase(s) and were phosphorylated at three sites in vi tro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phos phorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase wa s mainly in the membrane-associated fraction. These results suggest th at kinase(s) of the PKC family are specifically bound to and phosphory late Nef in vitro. The interaction of Nef with cellular kinases and it s phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.