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Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC - J. Exp. Med. (1997)

Bottom Line: We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation.Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor.These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

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Ly-49A function is impaired in me/me LAK cells. 9-d Ly-49A+  and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12  (C1498.Dd) and C1498 (H-2b) targets. D12 targets (A–F) and C1498 targets (G–L) were tested against Ly-49A+ effector cells (A–C and G–I) or  Ly-49A− effector cells (D–F and J–L) from +/+ (A, D, G, J), +/me (B,  E, H, K), or me/me mice (C, F, I, L). Assays were done in the absence of  antibody (open squares), or in the presence of anti–Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).
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Figure 7: Ly-49A function is impaired in me/me LAK cells. 9-d Ly-49A+ and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.Dd) and C1498 (H-2b) targets. D12 targets (A–F) and C1498 targets (G–L) were tested against Ly-49A+ effector cells (A–C and G–I) or Ly-49A− effector cells (D–F and J–L) from +/+ (A, D, G, J), +/me (B, E, H, K), or me/me mice (C, F, I, L). Assays were done in the absence of antibody (open squares), or in the presence of anti–Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).

Mentions: As shown in Fig. 7, Ly-49A function was significantly impaired in NK cells isolated from me/me mice, but, as in mev/mev mice, it was not completely absent. Ly-49A+ cells from wild-type +/+ or from heterozygous +/me were unable to lyse C1498.Dd targets (A and B), but Ly-49A+ cells from me/me mice were able to lyse these targets (C), albeit less effectively than Ly-49A− cells. Ly-49A− cells from all mice were able to lyse C1498.Dd targets equally well (Fig. 7, D–F). Addition of anti–Ly-49A mAb reversed Ly-49A–mediated inhibition of Ly-49A+ cells from all mice (Fig. 7, A–C), whereas control mAb had no effect. Ly-49A+ and Ly-49A− cells from all mice were able to lyse the H-2b target C1498 (Fig. 7, G–L), and addition of anti–Ly-49A or control mAb had no effect. These findings indicate that Ly-49A is functionally impaired in NK cells isolated from homozygous me/me mice. However, even in the complete absence of SHP-1, Ly-49A had some remaining inhibitory activity.


Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase.

Nakamura MC, Niemi EC, Fisher MJ, Shultz LD, Seaman WE, Ryan JC - J. Exp. Med. (1997)

Ly-49A function is impaired in me/me LAK cells. 9-d Ly-49A+  and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12  (C1498.Dd) and C1498 (H-2b) targets. D12 targets (A–F) and C1498 targets (G–L) were tested against Ly-49A+ effector cells (A–C and G–I) or  Ly-49A− effector cells (D–F and J–L) from +/+ (A, D, G, J), +/me (B,  E, H, K), or me/me mice (C, F, I, L). Assays were done in the absence of  antibody (open squares), or in the presence of anti–Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196152&req=5

Figure 7: Ly-49A function is impaired in me/me LAK cells. 9-d Ly-49A+ and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.Dd) and C1498 (H-2b) targets. D12 targets (A–F) and C1498 targets (G–L) were tested against Ly-49A+ effector cells (A–C and G–I) or Ly-49A− effector cells (D–F and J–L) from +/+ (A, D, G, J), +/me (B, E, H, K), or me/me mice (C, F, I, L). Assays were done in the absence of antibody (open squares), or in the presence of anti–Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).
Mentions: As shown in Fig. 7, Ly-49A function was significantly impaired in NK cells isolated from me/me mice, but, as in mev/mev mice, it was not completely absent. Ly-49A+ cells from wild-type +/+ or from heterozygous +/me were unable to lyse C1498.Dd targets (A and B), but Ly-49A+ cells from me/me mice were able to lyse these targets (C), albeit less effectively than Ly-49A− cells. Ly-49A− cells from all mice were able to lyse C1498.Dd targets equally well (Fig. 7, D–F). Addition of anti–Ly-49A mAb reversed Ly-49A–mediated inhibition of Ly-49A+ cells from all mice (Fig. 7, A–C), whereas control mAb had no effect. Ly-49A+ and Ly-49A− cells from all mice were able to lyse the H-2b target C1498 (Fig. 7, G–L), and addition of anti–Ly-49A or control mAb had no effect. These findings indicate that Ly-49A is functionally impaired in NK cells isolated from homozygous me/me mice. However, even in the complete absence of SHP-1, Ly-49A had some remaining inhibitory activity.

Bottom Line: We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation.Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor.These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Show MeSH
Related in: MedlinePlus