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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 inhibits phosphoinositide turnover in response to  H-2Dd target cells. RNK-mLy-49A.9 cells fail to generate InsP3 upon  stimulation with P388D1 targets. [3H]myoinositol-labeled RNK-16 and  RNK-mLy-49A.9 effectors (5 × 106 cells) were stimulated with 107 targets in a total volume of 1 ml at 37°C. Soluble InsP3 was resolved by ion  exchange chromatography. A brisk rise in InsP3 was seen in RNK-16  cells (left) in response to either YAC-1 (open squares) or P388D1 (H-2Dd)  (closed circles). Phosphoinositide turnover in RNK-mLy-49A.9 (right) was  stimulated by YAC-1, but not by P388D1.
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Figure 2: Ly-49A inhibits phosphoinositide turnover in response to H-2Dd target cells. RNK-mLy-49A.9 cells fail to generate InsP3 upon stimulation with P388D1 targets. [3H]myoinositol-labeled RNK-16 and RNK-mLy-49A.9 effectors (5 × 106 cells) were stimulated with 107 targets in a total volume of 1 ml at 37°C. Soluble InsP3 was resolved by ion exchange chromatography. A brisk rise in InsP3 was seen in RNK-16 cells (left) in response to either YAC-1 (open squares) or P388D1 (H-2Dd) (closed circles). Phosphoinositide turnover in RNK-mLy-49A.9 (right) was stimulated by YAC-1, but not by P388D1.

Mentions: Using the RNK-mLy-49A.9 cell line, we examined the inositol phosphate response following stimulation by YAC-1 (sensitive) and P388D1 (resistant) targets. Wild-type RNK-16, but not RNK-mLy-49A.9 cells, responded to the H-2Dd target P388D1 with a rapid increase in InsP3 at 2 min, as shown in Fig. 2. YAC-1, which is susceptible to lysis by both wild-type RNK-16 and RNK-mLy-49A.9, stimulates an InsP3 response in both cell types. The lack of phosphoinositide turnover in RNKmLy-49A.9 in response to H-2Dd target cells indicates that Ly-49A interrupts proximal signaling events in RNK-16.


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 inhibits phosphoinositide turnover in response to  H-2Dd target cells. RNK-mLy-49A.9 cells fail to generate InsP3 upon  stimulation with P388D1 targets. [3H]myoinositol-labeled RNK-16 and  RNK-mLy-49A.9 effectors (5 × 106 cells) were stimulated with 107 targets in a total volume of 1 ml at 37°C. Soluble InsP3 was resolved by ion  exchange chromatography. A brisk rise in InsP3 was seen in RNK-16  cells (left) in response to either YAC-1 (open squares) or P388D1 (H-2Dd)  (closed circles). Phosphoinositide turnover in RNK-mLy-49A.9 (right) was  stimulated by YAC-1, but not by P388D1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196152&req=5

Figure 2: Ly-49A inhibits phosphoinositide turnover in response to H-2Dd target cells. RNK-mLy-49A.9 cells fail to generate InsP3 upon stimulation with P388D1 targets. [3H]myoinositol-labeled RNK-16 and RNK-mLy-49A.9 effectors (5 × 106 cells) were stimulated with 107 targets in a total volume of 1 ml at 37°C. Soluble InsP3 was resolved by ion exchange chromatography. A brisk rise in InsP3 was seen in RNK-16 cells (left) in response to either YAC-1 (open squares) or P388D1 (H-2Dd) (closed circles). Phosphoinositide turnover in RNK-mLy-49A.9 (right) was stimulated by YAC-1, but not by P388D1.
Mentions: Using the RNK-mLy-49A.9 cell line, we examined the inositol phosphate response following stimulation by YAC-1 (sensitive) and P388D1 (resistant) targets. Wild-type RNK-16, but not RNK-mLy-49A.9 cells, responded to the H-2Dd target P388D1 with a rapid increase in InsP3 at 2 min, as shown in Fig. 2. YAC-1, which is susceptible to lysis by both wild-type RNK-16 and RNK-mLy-49A.9, stimulates an InsP3 response in both cell types. The lack of phosphoinositide turnover in RNKmLy-49A.9 in response to H-2Dd target cells indicates that Ly-49A interrupts proximal signaling events in RNK-16.

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