Physical and Functional Interaction of Nef with Lck

The nef gene is unique to the primate lentiviruses and encodes a cytoplasmic membrane-associated protein that affects T-cell signaling and is essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the perturbation of cell signaling by Nef in T-cells and found that Nef interacts with the T-cell restricted Lek tyrosine kinase both in vitro and in vivo. The molecular basis for this interaction was analyzed. We show that cell-derived Nef is precipitated in a synergistic manner by the recombinant Src homology 2 (SH2) and SH3 domains from Lck. A functional proline-rich motif and the tyrosine phosphorylation of Nef were evidenced as likely participants in this interaction. The precipitation of Nef by the Lck recombinant proteins was specific, since neither Fyn, Csk, p85 phosphatidylinositol 3-kinase nor phospholipase Cgamma SH2 domains coprecipitated Nef from T-cells. Finally, depressed Lck kinase activity resulted from the presence of Nef, both in vitro and in intact cells, and nef expression resulted in impairment of both proximal and distal Lck-mediated signaling events. These results provide a molecular basis for the Nef-induced T-cell signaling defect and its role in AIDS pathogenesis.