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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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Lysis of P388D1 (H-2Dd) cells is not altered in RNK-mLy49A/Y8F transfectants. Ly-49A expression on RNK transfectants was assessed by staining cells with either saline (dotted line) or FITC–anti-Ly-49A  (solid line). FACS® histograms show Ly-49A expression in wild-type  RNK-16 (B), RNK-mLy-49A.9 (D), RNK-mLy-49A/Y8F.1 (F) and  RNK-mLy-49A/Y8F.4 (H). Standard 4-h cytotoxicity assays were performed using P388D1 (H-2Dd) as targets. Effector cells were wild-type  RNK-16 (A), RNK-mLy-49A.9 (C), RNK-mLy-49A/Y8F.1 (E), or  RNK-mLy-49A/Y8F.4 (G). Effectors were preincubated with either media alone (open squares), anti-Ly-49A (closed circles), or isotype-matched  control antibody (anti-NK1.1, PK136, closed triangles), before addition of  targets.
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Figure 8: Lysis of P388D1 (H-2Dd) cells is not altered in RNK-mLy49A/Y8F transfectants. Ly-49A expression on RNK transfectants was assessed by staining cells with either saline (dotted line) or FITC–anti-Ly-49A (solid line). FACS® histograms show Ly-49A expression in wild-type RNK-16 (B), RNK-mLy-49A.9 (D), RNK-mLy-49A/Y8F.1 (F) and RNK-mLy-49A/Y8F.4 (H). Standard 4-h cytotoxicity assays were performed using P388D1 (H-2Dd) as targets. Effector cells were wild-type RNK-16 (A), RNK-mLy-49A.9 (C), RNK-mLy-49A/Y8F.1 (E), or RNK-mLy-49A/Y8F.4 (G). Effectors were preincubated with either media alone (open squares), anti-Ly-49A (closed circles), or isotype-matched control antibody (anti-NK1.1, PK136, closed triangles), before addition of targets.

Mentions: To examine further the role of SHP-1 in the function of Ly-49A, we mutated the tyrosine residue within the proposed SHP-1 binding motif in the Ly-49A cytoplasmic domain. RNK-16 cells transfected with this tyrosine mutant, (RNK-mLy-49A/Y8F) stained with anti– Ly-49A mAb at levels similar to those seen in RNK-mLy49A.9 (Fig. 8, D, F, H). Despite this level of Ly-49A expression, RNK-mLy-49A/Y8F clones 1 and 4, derived from separate transfections, were not inhibited in their capacity to lyse P388D1 (H-2Dd) cells (Fig. 8, E and G). Consistent with our earlier results, RNK-mLy-49A.9 cells could not lyse P388D1 (Fig. 8 C), whereas wild-type RNK-16 lysed P388D1 efficiently (A). Notably, neither anti–Ly-49A nor control mAb (anti–NK1.1) had any effect on lysis of P388D1 by RNK-mLy-49A/Y8F (Fig. 8, E and G), but anti–Ly-49A reversed the inhibition of P388D1 lysis by RNK-mLy-49A.9 (C). RNK-mLy-49A/Y8F clones 1 and 4 effectively lysed YAC-1 targets (data not shown). These data show that the tyrosine within the proposed immunoreceptor tyrosine-based inhibitory motif (ITIM) is required for the inhibitory effect of Ly-49A on RNK-16 cytotoxicity. These experiments complement the studies of motheaten mice, demonstrating a functional role for SHP-1 in the inhibitory activity of Ly-49A.


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)

Lysis of P388D1 (H-2Dd) cells is not altered in RNK-mLy49A/Y8F transfectants. Ly-49A expression on RNK transfectants was assessed by staining cells with either saline (dotted line) or FITC–anti-Ly-49A  (solid line). FACS® histograms show Ly-49A expression in wild-type  RNK-16 (B), RNK-mLy-49A.9 (D), RNK-mLy-49A/Y8F.1 (F) and  RNK-mLy-49A/Y8F.4 (H). Standard 4-h cytotoxicity assays were performed using P388D1 (H-2Dd) as targets. Effector cells were wild-type  RNK-16 (A), RNK-mLy-49A.9 (C), RNK-mLy-49A/Y8F.1 (E), or  RNK-mLy-49A/Y8F.4 (G). Effectors were preincubated with either media alone (open squares), anti-Ly-49A (closed circles), or isotype-matched  control antibody (anti-NK1.1, PK136, closed triangles), before addition of  targets.
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Figure 8: Lysis of P388D1 (H-2Dd) cells is not altered in RNK-mLy49A/Y8F transfectants. Ly-49A expression on RNK transfectants was assessed by staining cells with either saline (dotted line) or FITC–anti-Ly-49A (solid line). FACS® histograms show Ly-49A expression in wild-type RNK-16 (B), RNK-mLy-49A.9 (D), RNK-mLy-49A/Y8F.1 (F) and RNK-mLy-49A/Y8F.4 (H). Standard 4-h cytotoxicity assays were performed using P388D1 (H-2Dd) as targets. Effector cells were wild-type RNK-16 (A), RNK-mLy-49A.9 (C), RNK-mLy-49A/Y8F.1 (E), or RNK-mLy-49A/Y8F.4 (G). Effectors were preincubated with either media alone (open squares), anti-Ly-49A (closed circles), or isotype-matched control antibody (anti-NK1.1, PK136, closed triangles), before addition of targets.
Mentions: To examine further the role of SHP-1 in the function of Ly-49A, we mutated the tyrosine residue within the proposed SHP-1 binding motif in the Ly-49A cytoplasmic domain. RNK-16 cells transfected with this tyrosine mutant, (RNK-mLy-49A/Y8F) stained with anti– Ly-49A mAb at levels similar to those seen in RNK-mLy49A.9 (Fig. 8, D, F, H). Despite this level of Ly-49A expression, RNK-mLy-49A/Y8F clones 1 and 4, derived from separate transfections, were not inhibited in their capacity to lyse P388D1 (H-2Dd) cells (Fig. 8, E and G). Consistent with our earlier results, RNK-mLy-49A.9 cells could not lyse P388D1 (Fig. 8 C), whereas wild-type RNK-16 lysed P388D1 efficiently (A). Notably, neither anti–Ly-49A nor control mAb (anti–NK1.1) had any effect on lysis of P388D1 by RNK-mLy-49A/Y8F (Fig. 8, E and G), but anti–Ly-49A reversed the inhibition of P388D1 lysis by RNK-mLy-49A.9 (C). RNK-mLy-49A/Y8F clones 1 and 4 effectively lysed YAC-1 targets (data not shown). These data show that the tyrosine within the proposed immunoreceptor tyrosine-based inhibitory motif (ITIM) is required for the inhibitory effect of Ly-49A on RNK-16 cytotoxicity. These experiments complement the studies of motheaten mice, demonstrating a functional role for SHP-1 in the inhibitory activity of Ly-49A.

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