Limits...
Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity.

Brumbaugh KM, Binstadt BA, Billadeau DD, Schoon RA, Dick CJ, Ten RM, Leibson PJ - J. Exp. Med. (1997)

Bottom Line: Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function.Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk.These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT
Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

Show MeSH

Related in: MedlinePlus

MHC class I–bearing,  NK-resistant targets do not stimulate  an increase in Syk tyrosine phosphorylation. 107 NK cells were mixed  with 5 × 106 cells of (A) C1R or  C1R-B27, or (B) C1R or C1R-B7.  The reaction mixtures were pelleted  and incubated at 37°C for the indicated times. Syk immunoprecipitates  were resolved by SDS-PAGE, transferred to membrane, and probed  with antiphosphotyrosine mAb (P-tyr) or anti-Syk mAb (Syk).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199178&req=5

Figure 8: MHC class I–bearing, NK-resistant targets do not stimulate an increase in Syk tyrosine phosphorylation. 107 NK cells were mixed with 5 × 106 cells of (A) C1R or C1R-B27, or (B) C1R or C1R-B7. The reaction mixtures were pelleted and incubated at 37°C for the indicated times. Syk immunoprecipitates were resolved by SDS-PAGE, transferred to membrane, and probed with antiphosphotyrosine mAb (P-tyr) or anti-Syk mAb (Syk).

Mentions: Since our data suggest that Syk is functionally involved in the development of natural killing, it would follow that an NK-resistant target might not induce Syk tyrosine phosphorylation. Targets sensitive to NK lysis can be made resistant by transfection with an HLA class I molecule, provided the NK cell expresses a KIR specific for that class I (for review see reference 39). Since the parental target cell has only been altered by the addition of class I, we can use these cells to compare sensitive and resistant targets with the same triggering ligands. We compared NK-resistant, MHC class I–transfected C1R cells to the parental class I–deficient NK-sensitive C1R in their ability to induce Syk tyrosine phosphorylation. A DX9+ NK cell clone expressing the p70 immunoglobulin superfamily KIR specific for HLA-B27 (and related HLA-A and -B alleles) was stimulated with either C1R or C1R-B27 targets before lysing the cells and immunoprecipitating Syk. The sensitive C1R target stimulated an increase in Syk tyrosine phosphorylation, whereas stimulation with the resistant C1R-B27 did not result in a detectable change in Syk tyrosine phosphorylation (Fig. 8 A). This result was extended to include the CD94/ NKG2A/B inhibitory receptors of the C-type lectin superfamily. An NK cell clone expressing the CD94/NKG2A/B inhibitory receptor that can recognize HLA-B7 was stimulated with C1R or C1R-B7 targets before lysing and immunoprecipitating Syk. Again, the sensitive C1R target stimulated an increase in Syk tyrosine phosphorylation, whereas the resistant C1R-B7 target did not (Fig. 8 B). C1R-B27 and C1R-B7 have somewhat higher basal levels of tyrosine phosphorylated Syk than C1R (see B27 alone versus C1R alone, Fig. 8 A, B7 alone versus C1R alone, Fig. 8 B). However, increases in the tyrosine phosphorylation of NK cell–derived Syk were only seen after incubation with the NK-sensitive targets. These data suggest that the ability of a target to generate NK cell–mediated killing is associated with its capacity to stimulate Syk activation in NK cells.


Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity.

Brumbaugh KM, Binstadt BA, Billadeau DD, Schoon RA, Dick CJ, Ten RM, Leibson PJ - J. Exp. Med. (1997)

MHC class I–bearing,  NK-resistant targets do not stimulate  an increase in Syk tyrosine phosphorylation. 107 NK cells were mixed  with 5 × 106 cells of (A) C1R or  C1R-B27, or (B) C1R or C1R-B7.  The reaction mixtures were pelleted  and incubated at 37°C for the indicated times. Syk immunoprecipitates  were resolved by SDS-PAGE, transferred to membrane, and probed  with antiphosphotyrosine mAb (P-tyr) or anti-Syk mAb (Syk).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199178&req=5

Figure 8: MHC class I–bearing, NK-resistant targets do not stimulate an increase in Syk tyrosine phosphorylation. 107 NK cells were mixed with 5 × 106 cells of (A) C1R or C1R-B27, or (B) C1R or C1R-B7. The reaction mixtures were pelleted and incubated at 37°C for the indicated times. Syk immunoprecipitates were resolved by SDS-PAGE, transferred to membrane, and probed with antiphosphotyrosine mAb (P-tyr) or anti-Syk mAb (Syk).
Mentions: Since our data suggest that Syk is functionally involved in the development of natural killing, it would follow that an NK-resistant target might not induce Syk tyrosine phosphorylation. Targets sensitive to NK lysis can be made resistant by transfection with an HLA class I molecule, provided the NK cell expresses a KIR specific for that class I (for review see reference 39). Since the parental target cell has only been altered by the addition of class I, we can use these cells to compare sensitive and resistant targets with the same triggering ligands. We compared NK-resistant, MHC class I–transfected C1R cells to the parental class I–deficient NK-sensitive C1R in their ability to induce Syk tyrosine phosphorylation. A DX9+ NK cell clone expressing the p70 immunoglobulin superfamily KIR specific for HLA-B27 (and related HLA-A and -B alleles) was stimulated with either C1R or C1R-B27 targets before lysing the cells and immunoprecipitating Syk. The sensitive C1R target stimulated an increase in Syk tyrosine phosphorylation, whereas stimulation with the resistant C1R-B27 did not result in a detectable change in Syk tyrosine phosphorylation (Fig. 8 A). This result was extended to include the CD94/ NKG2A/B inhibitory receptors of the C-type lectin superfamily. An NK cell clone expressing the CD94/NKG2A/B inhibitory receptor that can recognize HLA-B7 was stimulated with C1R or C1R-B7 targets before lysing and immunoprecipitating Syk. Again, the sensitive C1R target stimulated an increase in Syk tyrosine phosphorylation, whereas the resistant C1R-B7 target did not (Fig. 8 B). C1R-B27 and C1R-B7 have somewhat higher basal levels of tyrosine phosphorylated Syk than C1R (see B27 alone versus C1R alone, Fig. 8 A, B7 alone versus C1R alone, Fig. 8 B). However, increases in the tyrosine phosphorylation of NK cell–derived Syk were only seen after incubation with the NK-sensitive targets. These data suggest that the ability of a target to generate NK cell–mediated killing is associated with its capacity to stimulate Syk activation in NK cells.

Bottom Line: Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function.Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk.These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT
Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

Show MeSH
Related in: MedlinePlus