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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 mev/mev LAK cells. 9-d Ly-49A+  and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12  (C1498.Dd) targets against Ly-49A+ effector cells (A–C) or Ly-49A− effector cells (D–F) from +/+ (A and D), +/mev (B and E), or mev/mev mice  (C and F). 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 5: Ly-49A function is impaired in mev/mev LAK cells. 9-d Ly-49A+ and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.Dd) targets against Ly-49A+ effector cells (A–C) or Ly-49A− effector cells (D–F) from +/+ (A and D), +/mev (B and E), or mev/mev mice (C and F). 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: In each of four experiments, with homozygous mev/mev mice the function of Ly-49A was impaired in that C1498.Dd targets were lysed by Ly-49A+ effectors, albeit less effectively than by Ly-49A− effectors. However, Ly-49A remained partially effective in cells from mev/mev mice in that addition of anti–Ly-49A still increased lysis of the C1498.Dd targets. Fig. 5 shows a representative experiment in which Ly-49A+ cells from wild-type C57BL/6 +/+ or from heterozygous +/mev mice were unable to lyse C1498.Dd targets (A and B), but Ly-49A+ cells from homozygous mice were able to lyse these targets (C). Ly-49A− cells from all mice were able to lyse C1498.Dd equally well (Fig. 5, D–F). Addition of anti–Ly-49A mAb reversed the Ly-49A–mediated inhibition to levels similar to those of Ly-49A− cells from all mice, whereas isotype-matched control anti-gp42 mAb had no effect. Ly-49A+ and Ly-49A− cells from all mice were able to lyse the H-2b target C1498 and addition of mAb anti–Ly-49A or anti-gp42 had no effect (data not shown). These findings indicate that the function of Ly49A is partially impaired in IL-2–activated NK cells isolated from homozygous mev/mev mice.


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 mev/mev LAK cells. 9-d Ly-49A+  and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12  (C1498.Dd) targets against Ly-49A+ effector cells (A–C) or Ly-49A− effector cells (D–F) from +/+ (A and D), +/mev (B and E), or mev/mev mice  (C and F). 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).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Ly-49A function is impaired in mev/mev LAK cells. 9-d Ly-49A+ and Ly-49A− LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.Dd) targets against Ly-49A+ effector cells (A–C) or Ly-49A− effector cells (D–F) from +/+ (A and D), +/mev (B and E), or mev/mev mice (C and F). 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: In each of four experiments, with homozygous mev/mev mice the function of Ly-49A was impaired in that C1498.Dd targets were lysed by Ly-49A+ effectors, albeit less effectively than by Ly-49A− effectors. However, Ly-49A remained partially effective in cells from mev/mev mice in that addition of anti–Ly-49A still increased lysis of the C1498.Dd targets. Fig. 5 shows a representative experiment in which Ly-49A+ cells from wild-type C57BL/6 +/+ or from heterozygous +/mev mice were unable to lyse C1498.Dd targets (A and B), but Ly-49A+ cells from homozygous mice were able to lyse these targets (C). Ly-49A− cells from all mice were able to lyse C1498.Dd equally well (Fig. 5, D–F). Addition of anti–Ly-49A mAb reversed the Ly-49A–mediated inhibition to levels similar to those of Ly-49A− cells from all mice, whereas isotype-matched control anti-gp42 mAb had no effect. Ly-49A+ and Ly-49A− cells from all mice were able to lyse the H-2b target C1498 and addition of mAb anti–Ly-49A or anti-gp42 had no effect (data not shown). These findings indicate that the function of Ly49A is partially impaired in IL-2–activated NK cells isolated from homozygous mev/mev mice.

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