Physical and Functional Interaction of Nef with Lck

Abstract

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.

The HIV Nef Protein Alters Ca2+ Signaling in Myelomonocytic Cells through SH3-mediated Protein-Protein Interactions

“…Instead, thapsigargin efficiently depleted intracellular Ca 2ϩ stores both in nef-expressing and in control cells (Fig. 3, E and F (14,(22)(23)(24)(25) with a high affinity via interactions between a prolinerich motif in the Nef core and SH3 domains of Src-like PTKs. The mutation of the Nef proline motifs prevents the binding of Nef to Src-like PTKs and their subsequent activation but does not abolish other Nef effects such as cell surface CD4 downregulation (Fig.…”

“…In particular, studies in vitro with recombinant proteins and crystal structure determinations have shown that the core region of Nef, via a proline-rich motif, binds with a high affinity to the SH3 domains of Src-like protein-tyrosine kinases (Src-like PTKs) (14,22). Four members of the Src kinases family, p56 lck , p56/59 hck , p59 fyn , and p53 /56 lyn have been shown to be able to interact physically with Nef (14,(22)(23)(24)(25). Interestingly, the affinity of Nef-p56/59 hck interaction is the highest known for an SH3-mediated proteinprotein binding (23).…”

Biochemical mechanisms of HIV induced T cell apoptosis

“…The JAK3/STAT5 signaling pathway has been shown to upregulate anti-apoptotic proteins, such as Bcl-2 and BclxL. 31,32 These data suggest that HIV mediated inhibition of Increased FasL expression 85 Binding of p53, Hck, pp44 MAPK/ERK1 83,136 Decreased expression of IL-2Ra chain 83,135 Inhibition of proliferation in response to IL-2 83 Decreased MHC class I surface expression 86,133 Another function of Vpr is cell cycle arrest. Relevant to our subsequent discussion of HIV-induced death of bystander cells, Vpr induces cell cycle arrest in both infected and uninfected cells.…”

Using death to one's advantage: HIV modulation of apoptosis

“…Nef associates with cellular serine/threonine kinases (possibly PAK), as well as src-like tyrosine kinases (Hck and Lck). [136][137][138] Both of these types of kinases are important in T cell activation. [136][137][138][139] Consistent with this observation, Fas-L is expressed following activation of the TCR leading to the phosphorylation of these kinases and expression is inhibited by cell treatment with immunosuppressive drugs.…”

“…[136][137][138] Both of these types of kinases are important in T cell activation. [136][137][138][139] Consistent with this observation, Fas-L is expressed following activation of the TCR leading to the phosphorylation of these kinases and expression is inhibited by cell treatment with immunosuppressive drugs. 139 Ras signaling has also been implicated in tyrosine kinase activation and hence both the Ras and Lck pathways may contribute to the up-regulation of Fas-L transcription.…”