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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 an  early rise in tyrosine phosphorylation induced by target cell  stimulation. Tyrosine phosphorylation of proteins in RNK-16  cells stimulated with P388D1  cells is shown on the left side of  the figure. RNK-mLy-49A.9  cells stimulated with P388D1  cells are in the center, and  RNK-mLy-49A.9 cells stimulated with YAC-1 cells are on  the right. Target cell stimulation  time points were 0, 0.5, 1.0, and  5.0 min. RNK-16 and RNKmLy-49A.9 effector cells were  metabolically labeled with 32Porthophosphate. After washing,  107 labeled effector cells and 107  unlabeled target cells were stimulated in a total volume of 1 ml  complete phosphate-free RPMI  with a brief 50 g contact spin,  followed by incubation at 37°C  for the indicated time. Cells  were then immediately lysed in  cold HNTG buffer with 1% Triton X-100. Clarifed precleared  cell lysates were immunoprecipitated with APT (4G10),  washed, and resolved by reducing 8% SDS-PAGE. Gels were  dried and developed by autoradiography.
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Figure 3: Ly-49A inhibits an early rise in tyrosine phosphorylation induced by target cell stimulation. Tyrosine phosphorylation of proteins in RNK-16 cells stimulated with P388D1 cells is shown on the left side of the figure. RNK-mLy-49A.9 cells stimulated with P388D1 cells are in the center, and RNK-mLy-49A.9 cells stimulated with YAC-1 cells are on the right. Target cell stimulation time points were 0, 0.5, 1.0, and 5.0 min. RNK-16 and RNKmLy-49A.9 effector cells were metabolically labeled with 32Porthophosphate. After washing, 107 labeled effector cells and 107 unlabeled target cells were stimulated in a total volume of 1 ml complete phosphate-free RPMI with a brief 50 g contact spin, followed by incubation at 37°C for the indicated time. Cells were then immediately lysed in cold HNTG buffer with 1% Triton X-100. Clarifed precleared cell lysates were immunoprecipitated with APT (4G10), washed, and resolved by reducing 8% SDS-PAGE. Gels were dried and developed by autoradiography.

Mentions: To examine the effect of Ly-49A–mediated inhibition on protein tyrosine phosphorylation, [32P]orthophosphate-labeled RNK-16 and RNK-mLy-49A.9 cells were stimulated with YAC-1 (H-2a) and P388D1 (H-2Dd) target cells. Because Ly-49A interrupts InsP3 turnover, an early signaling event, we examined the effect of target stimulation at 30 s, 1 min, and 5 min time points. Lysates from cells stimulated for these time intervals were immunoprecipitated with APT (4G10) and resolved by SDS-PAGE (Fig. 3). RNK-16 cells stimulated with P388D1 (H-2Dd) showed a rapid increase in protein tyrosine phosphorylation at 30 s to 1 min, which diminished toward basal levels at 5 min (Fig. 3, left). In contrast, RNK-mLy-49A.9 cells stimulated with P388D1 failed to show an increase in protein tyrosine phosphorylation at 30 s to 1 min, but showed a minimal increase at 5 min (Fig. 3, center). Nonetheless, RNK-mLy-49A.9 cells demonstrated rapid protein tyrosine phosphorylation in response to YAC-1 after 30 s, demonstrating that this signaling pathway was intact (Fig. 3, right). Wild-type RNK-16 showed a similar response to YAC-1 target cells (data not shown). Thus, susceptible, but not resistant, targets induce very brisk increases in protein tyrosine phosphorylation, and mouse Ly-49A specifically interrupts rapid tyrosine phosphorylation in response to P388D1 (H-2Dd) targets.


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 an  early rise in tyrosine phosphorylation induced by target cell  stimulation. Tyrosine phosphorylation of proteins in RNK-16  cells stimulated with P388D1  cells is shown on the left side of  the figure. RNK-mLy-49A.9  cells stimulated with P388D1  cells are in the center, and  RNK-mLy-49A.9 cells stimulated with YAC-1 cells are on  the right. Target cell stimulation  time points were 0, 0.5, 1.0, and  5.0 min. RNK-16 and RNKmLy-49A.9 effector cells were  metabolically labeled with 32Porthophosphate. After washing,  107 labeled effector cells and 107  unlabeled target cells were stimulated in a total volume of 1 ml  complete phosphate-free RPMI  with a brief 50 g contact spin,  followed by incubation at 37°C  for the indicated time. Cells  were then immediately lysed in  cold HNTG buffer with 1% Triton X-100. Clarifed precleared  cell lysates were immunoprecipitated with APT (4G10),  washed, and resolved by reducing 8% SDS-PAGE. Gels were  dried and developed by autoradiography.
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Related In: Results  -  Collection

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Figure 3: Ly-49A inhibits an early rise in tyrosine phosphorylation induced by target cell stimulation. Tyrosine phosphorylation of proteins in RNK-16 cells stimulated with P388D1 cells is shown on the left side of the figure. RNK-mLy-49A.9 cells stimulated with P388D1 cells are in the center, and RNK-mLy-49A.9 cells stimulated with YAC-1 cells are on the right. Target cell stimulation time points were 0, 0.5, 1.0, and 5.0 min. RNK-16 and RNKmLy-49A.9 effector cells were metabolically labeled with 32Porthophosphate. After washing, 107 labeled effector cells and 107 unlabeled target cells were stimulated in a total volume of 1 ml complete phosphate-free RPMI with a brief 50 g contact spin, followed by incubation at 37°C for the indicated time. Cells were then immediately lysed in cold HNTG buffer with 1% Triton X-100. Clarifed precleared cell lysates were immunoprecipitated with APT (4G10), washed, and resolved by reducing 8% SDS-PAGE. Gels were dried and developed by autoradiography.
Mentions: To examine the effect of Ly-49A–mediated inhibition on protein tyrosine phosphorylation, [32P]orthophosphate-labeled RNK-16 and RNK-mLy-49A.9 cells were stimulated with YAC-1 (H-2a) and P388D1 (H-2Dd) target cells. Because Ly-49A interrupts InsP3 turnover, an early signaling event, we examined the effect of target stimulation at 30 s, 1 min, and 5 min time points. Lysates from cells stimulated for these time intervals were immunoprecipitated with APT (4G10) and resolved by SDS-PAGE (Fig. 3). RNK-16 cells stimulated with P388D1 (H-2Dd) showed a rapid increase in protein tyrosine phosphorylation at 30 s to 1 min, which diminished toward basal levels at 5 min (Fig. 3, left). In contrast, RNK-mLy-49A.9 cells stimulated with P388D1 failed to show an increase in protein tyrosine phosphorylation at 30 s to 1 min, but showed a minimal increase at 5 min (Fig. 3, center). Nonetheless, RNK-mLy-49A.9 cells demonstrated rapid protein tyrosine phosphorylation in response to YAC-1 after 30 s, demonstrating that this signaling pathway was intact (Fig. 3, right). Wild-type RNK-16 showed a similar response to YAC-1 target cells (data not shown). Thus, susceptible, but not resistant, targets induce very brisk increases in protein tyrosine phosphorylation, and mouse Ly-49A specifically interrupts rapid tyrosine phosphorylation in response to P388D1 (H-2Dd) targets.

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