Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts - PubMed (original) (raw)

Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts

Carsten Watzl et al. J Exp Med. 2003.

Abstract

A dynamic balance of positive and negative signals regulates target cell lysis by natural killer (NK) cells upon engagement of a variety of different activation receptors and of inhibitory receptors that recruit the tyrosine phosphatase SHP-1. However, the step at which activation signals are blocked by SHP-1 is not known. We have been using activation receptor 2B4 (CD244) to study the influence of inhibitory receptors on NK cell activation. Engagement of inhibitory receptors by HLA class I on target cells blocks phosphorylation of 2B4, placing the inhibitory step at the level, or upstream of 2B4 phosphorylation. Here we show that phosphorylated 2B4, after engagement with either antibodies or target cells that express the 2B4 ligand, is found exclusively in a detergent-resistant membrane fraction that contains lipid rafts. Integrity of lipid rafts was essential for phosphorylation and activating function of 2B4. Coengagement of inhibitory receptors blocked 2B4 phosphorylation and 2B4 association with detergent-resistant membranes, indicating that inhibitory receptors function upstream of raft-dependent signals. Recruitment of 2B4 into detergent-resistant membrane fractions and 2B4 phosphorylation were dependent on actin polymerization. Blocking actin cytoskeleton-dependent raft recruitment of different receptors may be a general mechanism by which inhibitory receptors control NK cell activation.

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Figures

Figure 1.

Figure 1.

Phosphorylated 2B4 is found exclusively in DRM domains. (A) The CD16-negative human NK cell line YTS was stimulated with antibodies against CD56 or 2B4 that were cross-linked with a secondary antibody for 5 min at 37°C. Subsequently, cells were lysed and DRM isolated by sucrose gradient centrifugation. Neighboring fractions were combined and analyzed by Western blotting using antibodies against 2B4 (top panel), CD45 (middle panel), or HRP-conjugated cholera toxin B-subunit (CTx, bottom panel). CD45 is a marker for the soluble fraction (10/11) while CTx marks the DRM fraction (4/5). (B) YTS cells were stimulated with an antibody against 2B4 and cross-linked with a mixture of unlabeled and HRP-conjugated secondary antibody for the indicated time at 37°C. After DRM isolation the different fractions were analyzed for the presence of HRP activity using a colorimetric substrate assay followed by photometric analysis. (C) The DRM (4/5) and the soluble fractions (10/11) from the samples shown in panel A were immunoprecipitated using an anti-2B4 antibody and analyzed by anti-phosphotyrosine Western blotting.

Figure 2.

Figure 2.

Target cell contact induces recruitment of 2B4 into DRM domains. Purified polyclonal human NK cells and 721.221 cells were either incubated separately (0′) or mixed (5′) for 5 min at 37°C. After cell lysis, DRM was isolated, neighboring fractions were combined and immunoprecipitated using a control antibody (IgG1) followed by anti-2B4 immunoprecipitation (2B4). Samples were analyzed by anti-2B4 Western blotting (2B4) and reblotted using an anti-phosphotyrosine antibody (2B4-P). DRM isolation was monitored by analyzing the different fraction using an anti-CD45 antibody and HRP-conjugated cholera toxin B-subunit (CTx).

Figure 3.

Figure 3.

Lipid rafts are essential for NK cell activation and 2B4 function. (A and B) Purified polyclonal human NK cells were control-treated or cholesterol-depleted using MCD and analyzed either immediately or after a 3 h recovery in complete medium. Cells were analyzed in a 51Cr-release assay using 721.221 target cells (A) or in a redirected lysis assay using P815 target cells (B) coated with control antibody (open symbols) or anti-2B4 antibody (filled symbols).

Figure 4.

Figure 4.

Lipid rafts are essential for 2B4 phosphorylation. (A) The NK cell line YTS was treated with MCD as described in Fig. 3 and stimulated with a control antibody (CD56) or an anti-2B4 antibody and analyzed as described in Fig. 1 A (top panel), and reblotted with an anti-phosphotyrosine antibody (middle panel). DRM isolation was monitored by analyzing the different fraction using an anti-CD45 antibody (unpublished data) and HRP-conjugated cholera toxin B-subunit (bottom panel). (B) The DRM (4/5) and the soluble fractions (10/11) from the samples shown in panel A were immunoprecipitated using an anti-2B4 antibody and analyzed by anti-phosphotyrosine Western blotting.

Figure 5.

Figure 5.

Accumulation of phosphorylated 2B4 outside of DRM domains after pervanadate treatment. The NK cell line YTS was treated with PBS (−) or 100 μM pervanadate (+) in PBS for 10 min at 37°C and DRM was isolated. The DRM fraction (4/5) and the detergent soluble fraction (10/11) were immunoprecipitated with a control antibody (IgG1) followed by anti-2B4 immunoprecipitation. Samples were analyzed by Western blotting using an rabbit anti-2B4 antibody (top panel) and reblotted with an anti-phosphotyrosine antibody (bottom panel).

Figure 6.

Figure 6.

KIR2DL1 blocks DRM recruitment and phosphorylation of 2B4. YTS-2DL1 cells and 221-Cw3 or 221-Cw4 cells were either incubated separately (0′) or together (5′) for 5 min at 37°C. After cell lysis DRM was isolated, neighboring fractions were combined and analyzed by Western blotting using antibodies against 2B4 (top panel) or KIR2DL1 (bottom panel). DRM isolation was monitored by analyzing the different fraction using an anti-CD45 antibody and HRP-conjugated cholera toxin B-subunit as in Figs. 1 and 2 (unpublished data). (B) The DRM (4/5) and the soluble fractions (10/11) from the samples shown in panel A were immunoprecipitated (IP) using a control antibody (IgG1) followed by an anti-2B4 antibody (2B4) and analyzed by anti-2B4 Western blotting (2B4) and reblotted using an anti-phosphotyrosine antibody (2B4-P).

Figure 7.

Figure 7.

Recruitment of 2B4 to DRM domains is dependent on Src-family kinases and on actin polymerization. The CD16-negative human NK cell line YTS was treated for 30 min at 37°C with carrier only (DMSO) or the indicated inhibitors. Subsequently, cells were stimulated by an anti-2B4 antibody and analyzed as described in Fig. 1 B. Gradient fractions from DMSO- and Cytochalasin D–treated cells were also analyzed by Western blotting which confirmed the result for the distribution of 2B4 and demonstrated that Cytochalasin D treatment does not affect the integrity and isolation of DRM fractions (unpublished data).

Figure 8.

Figure 8.

2B4 phosphorylation is dependent on the actin cytoskeleton. (A) Purified polyclonal human NK cells were treated for 30 min at 37°C with carrier only (DMSO) or the indicated inhibitors. Subsequently, cells were either incubated separately (0′) or after mixing with 721.221 cells (5′) for 5 min at 37°C. After lysis, samples were immunoprecipitated using a control antibody (IgG1, shown is the DMSO-treated sample after target cell mixing and 5′ incubation) followed by an anti-2B4 antibody. Immunoprecipitates were analyzed by anti-phosphotyrosine Western blotting (2B4-P) and reblotted using an anti-2B4 antibody (2B4). (B) The same cells described in panel A were treated with pervanadate, lysed, immunoprecipitated, and analyzed as described for panel A.

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