Activation of the PAK-related kinase by human immunodeficiency virus type 1 Nef in primary human peripheral blood lymphocytes and macrophages leads to phosphorylation of a PIX-p95 complex

Abstract

Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication in both primary T lymphocytes and monocyte-derived macrophages. This enhancement phenotype has been linked to the ability of Nef to modulate the activity of cellular kinases. We find that despite the reported high-affinity interaction between Nef and the Src kinase Hck in vitro, a Nef-Hck interaction in the context of HIV-1-infected primary macrophages is not detectable. However, Nef binding and activation of the PAK-related kinase and phosphorylation of its substrate could be readily detected in both infected primary T lymphocytes and macrophages. Furthermore, we show that this substrate is a complex composed of the recently characterized PAK interacting partner PIX (PAK-interacting guanine nucleotide exchange factor) and its tightly associated p95 protein. PAK and PIX-p95 appear to be differentially activated and phosphorylated depending on the intracellular environment in which nef is expressed. These results identify the PIX-p95 complex as a novel effector of Nef in primary cells and suggest that the regulation of the PAK signaling pathway may differ in T cells and macrophages.

FIG. 1

(A) Replication kinetics of SF162 and R25 in stimulated and resting PBMCs. PBMCs were stimulated with IL-2 and PHA before infection (stimulated) or infected and then stimulated 4 days postinfection (resting). The amount of p24 capsid antigen released into the culture supernatant was quantitated every 3 to 4 days over periods of 14 and 25 days, respectively, for infected macrophages and PBMCs. □, SF162 wild type; ⧫, SF162 Δnef clone 9; ■, SF162 Δnef clone 10; ○, R25 wild type; ●, R25 Δnef clone 3; ▵, R25 Δnef clone 14. (B) Replication kinetics of SF162 and R25 in MDM. MDM were infected on day 10 postisolation with 40 ng of virus p24. □, SF162 wild type; ⧫, SF162 Δnef clone 9; ■, SF162 Δnef clone 10; ○, R25 wild type; ●, R25 Δnef clone 3; ▵, R25 Δnef clone 14.

FIG. 2

(A) MDM were grown and infected in medium with serum and harvested as described in Materials and Methods. Approximately 2 × 10⁵ to 5 × 10⁵ cells were used for each immunoprecipitation. Western analysis was performed with an anti-Hck antibody (Anti-Hck) (top row), and the blot was probed with anti-phosphotyrosine antibody (Anti-pTyr) (bottom row). Hck immunoprecipitated from mock-infected cells (lane 1) served as a control for the level of Hck protein present. The two forms of Hck protein are due to alternative translation initiation start sites giving rise to proteins of 59 and 61 kDa. The top edge of a band visible at 49.4 kDa is the immunoglobulin heavy chain. The amount of Nef protein immunoprecipitated was also determined and is shown in Fig. 3, lanes 5 and 6. Anti-Nef IPs from mock-infected cells (lane 2), SF162 wild type-infected cells (lane 3), and R25 wild type-infected cells (lane 4) are shown. (B) IVKAs were performed at 3, 5, and 7 days p.i. on Hck IPs from mock-infected (lanes 1, 3, and 5, respectively) and SF162 wild type-infected (lanes 2, 4, and 6, respectively) macrophages. The reactions were run on SDS–10% PAGE gels and dried on Whatman 3 MM filter paper, and nucleotide incorporation was detected by phosphorimager analysis. The levels of Nef expression were determined by immunoprecipitation and Western analyses.

FIG. 3

(A) Nef IPs from macrophages (lanes 1 to 6) infected and cultured in the presence (+) or absence (−) of serum for 10 days were subjected to an IVKA. IVKAs of Nef IPs from the chronically infected T-cell line CEMx174 (lanes 7 to 9) were run side by side with the IPs from macrophages for comparison. Nef IVKAs were performed on infected PBMCs at day 12 p.i. (lanes 10 to 13). Results for mock-infected cells (lanes 1, 4, and 10) and cells infected with SF162 wild type (lanes 2, 5, and 12), SF162 Δnef (lane 11), SF162 Nef P71A (lane 13), R25 wild type (lanes 3 and 6), SF2 wild type (lane 7), SF2 Nef P73A (lane 8), or SF2 Δnef (lane 9) are shown. Underneath, the levels of Nef protein present in each IP as detected by Western analysis are shown. (B) PAK IPs from macrophages infected and cultured in the presence (+) or absence (−) of serum were subjected to IVKAs. Results for mock-infected cells (lanes 1 and 4), SF162 wild type-infected cells (lanes 2 and 5) and R25 wild type-infected cells (lanes 3 and 6) are shown. Results of IVKAs of PAK IPs from mock-infected (lane 7) and chronically SF2-infected (lane 8) CEMx174 cells are shown for comparison. The band at 49.4 is the immunoglobulin heavy chain.

FIG. 4

(A) Reimmunoprecipitation of PAK and PIX from Nef IPs of infected CEMx174 cells. Cell lysates from ∼7 × 10⁶ cells were split into two portions (2.5 × 10⁶ and 4.5 × 10⁶ cells) and subjected to Nef immunoprecipitation and IVKA. One portion was reserved as the IVKA control for SF2 Δnef (Nef−) (lane 1), SF2 wild type (SF2) (lane 2) and SF2 Nef P73A (P73) (lane 3). The larger portion was subjected to reimmunoprecipitation with anti-PAK (P) (lanes 4, 7, and 11), anti-PIX (X) (lanes 5, 8, and 12), anti-Vav (V) (lanes 6, 9, and 13), or anti-Hck (H) (lane 10) antibodies as described in Materials and Methods. Western analysis with either anti-PAK (P) (lane 14), anti-PIX (X) (lane 15), or anti-Vav (V) (lane 16) antibody was performed on CEMx174 cell lysates. The band at 49.4 kDa is the immunoglobulin heavy chain. (B) Reimmunoprecipitation of PAK and PIX from infected primary macrophages was performed as described above except that ∼1 × 10⁶ cells were used. Results of IVKAs performed as controls for SF162 Δnef-infected (Nef−), SF162 wild-type (SF162), and R25 wild-type (R25) cells are shown in lanes 1, 2, and 3, respectively. Reimmunoprecipitation was performed with anti-PAK (lanes 4, 7, and 10), anti-PIX (lanes 5, 8, and 11), or anti-Vav (lanes 6, 9, and 12) antibodies. Western analysis with anti-PIX, anti-PAK, or anti-Vav antibody was performed on macrophage cell lysates, and results are shown in lanes 13, 14, and 15, respectively.