RT Journal Article SR Electronic(1) A1 Bodéus, Monique A1 Marie-Cardine, Anne A1 Bougeret, Cécile A1 Ramos-Morales, Francisco A1 Benarous, RichardYR 1995 T1 In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase JF Journal of General Virology, VO 76 IS 6 SP 1337 OP 1344 DO https://doi.org/10.1099/0022-1317-76-6-1337 PB Microbiology Society, SN 1465-2099, AB Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: 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-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis., UL https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-76-6-1337