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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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The tyrosine phosphatase SHP-1 associates with  mouse Ly-49A in pervanadatestimulated RNK-16 cells. 1.5 ×  107 unstimulated or pervanadatestimulated RNK-16 cells or  RNK-mLy-49A.9 cells were incubated in complete RPMI for 5  min at 37°C, washed, and lysed  in cold HNTG lysis buffer containing 1% Triton X-100. Clarified  precleared lysates were immunoprecipitated with anti–Ly-49A  (A1, lanes 5–8) or isotypematched control mAb (NK1.1,  PK136, lanes 1–4), washed, and  resolved by 8% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF  membranes, immunoblotted with  anti-SHP-1 antiserum, and developed with 125I–protein A followed by autoradiography.
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Figure 4: The tyrosine phosphatase SHP-1 associates with mouse Ly-49A in pervanadatestimulated RNK-16 cells. 1.5 × 107 unstimulated or pervanadatestimulated RNK-16 cells or RNK-mLy-49A.9 cells were incubated in complete RPMI for 5 min at 37°C, washed, and lysed in cold HNTG lysis buffer containing 1% Triton X-100. Clarified precleared lysates were immunoprecipitated with anti–Ly-49A (A1, lanes 5–8) or isotypematched control mAb (NK1.1, PK136, lanes 1–4), washed, and resolved by 8% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF membranes, immunoblotted with anti-SHP-1 antiserum, and developed with 125I–protein A followed by autoradiography.

Mentions: In other lymphoid cells, SHP-1 has been shown to interrupt early tyrosine phosphorylation events. The mouse Ly-49A cytoplasmic domain includes the proposed SHP-1 binding motif VxYxxV (21, 22). These structural features of Ly-49A, and the results obtained in Fig. 3, suggested that Ly-49A– dependent inhibition of NK cell function might be mediated by SHP-1. Therefore, we first investigated the binding of SHP-1 to Ly-49A in RNK-16 cells by performing immunoprecipitation experiments. RNK-16 and RNK-mLy49A.9 cells were stimulated with pervanadate, a phosphatase inhibitor that pharmacologically increases protein tyrosine phosphorylation (34). Lysates from stimulated and unstimulated cells were precipitated with anti–Ly-49A, and precipitates were examined for the presence of Ly-49A–associated SHP-1 by Western blot analysis. As shown in Fig. 4, SHP-1 was present only in anti–Ly-49A immunoprecipitates from pervanadate-stimulated RNK-mLy-49A.9 cells. SHP-1 was not detected in anti–Ly-49A immunoprecipitates from unstimulated RNK-mLy-49A.9 cells or in isotype-matched control mAb immunoprecipitates from unstimulated or stimulated RNK-mLy-49A.9 cells. SHP-1 was not detected in anti–Ly-49A or control mAb immunoprecipitates from RNK-16 cells, regardless of stimulation.


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)

The tyrosine phosphatase SHP-1 associates with  mouse Ly-49A in pervanadatestimulated RNK-16 cells. 1.5 ×  107 unstimulated or pervanadatestimulated RNK-16 cells or  RNK-mLy-49A.9 cells were incubated in complete RPMI for 5  min at 37°C, washed, and lysed  in cold HNTG lysis buffer containing 1% Triton X-100. Clarified  precleared lysates were immunoprecipitated with anti–Ly-49A  (A1, lanes 5–8) or isotypematched control mAb (NK1.1,  PK136, lanes 1–4), washed, and  resolved by 8% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF  membranes, immunoblotted with  anti-SHP-1 antiserum, and developed with 125I–protein A followed by autoradiography.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196152&req=5

Figure 4: The tyrosine phosphatase SHP-1 associates with mouse Ly-49A in pervanadatestimulated RNK-16 cells. 1.5 × 107 unstimulated or pervanadatestimulated RNK-16 cells or RNK-mLy-49A.9 cells were incubated in complete RPMI for 5 min at 37°C, washed, and lysed in cold HNTG lysis buffer containing 1% Triton X-100. Clarified precleared lysates were immunoprecipitated with anti–Ly-49A (A1, lanes 5–8) or isotypematched control mAb (NK1.1, PK136, lanes 1–4), washed, and resolved by 8% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF membranes, immunoblotted with anti-SHP-1 antiserum, and developed with 125I–protein A followed by autoradiography.
Mentions: In other lymphoid cells, SHP-1 has been shown to interrupt early tyrosine phosphorylation events. The mouse Ly-49A cytoplasmic domain includes the proposed SHP-1 binding motif VxYxxV (21, 22). These structural features of Ly-49A, and the results obtained in Fig. 3, suggested that Ly-49A– dependent inhibition of NK cell function might be mediated by SHP-1. Therefore, we first investigated the binding of SHP-1 to Ly-49A in RNK-16 cells by performing immunoprecipitation experiments. RNK-16 and RNK-mLy49A.9 cells were stimulated with pervanadate, a phosphatase inhibitor that pharmacologically increases protein tyrosine phosphorylation (34). Lysates from stimulated and unstimulated cells were precipitated with anti–Ly-49A, and precipitates were examined for the presence of Ly-49A–associated SHP-1 by Western blot analysis. As shown in Fig. 4, SHP-1 was present only in anti–Ly-49A immunoprecipitates from pervanadate-stimulated RNK-mLy-49A.9 cells. SHP-1 was not detected in anti–Ly-49A immunoprecipitates from unstimulated RNK-mLy-49A.9 cells or in isotype-matched control mAb immunoprecipitates from unstimulated or stimulated RNK-mLy-49A.9 cells. SHP-1 was not detected in anti–Ly-49A or control mAb immunoprecipitates from RNK-16 cells, regardless of stimulation.

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