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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.

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32P incorporation into NK cell–derived Syk. 107 32P-labeled  NK cells were mixed with 5 × 106 cells of the indicated target (K562 or  C1R), pelleted, and incubated at 37°C for the indicated times. Syk immunoprecipitates were resolved by SDS-PAGE, transferred to membrane, and exposed to x-ray film (Syk) followed by probing with antiphosphotyrosine mAb (P-tyr blot). cpm from phosphoamino acid analysis  were: NK + K562 — 5′: 2.3-fold increase in [32P]Tyr, 2-fold increase in  [32P]Ser; NK + K562 — 10′: 1.5-fold increase in [32P]Tyr, 1.5-fold increase in [32P]Ser; NK + C1R — 5′: 2.4-fold increase in [32P]Tyr, 1.6-fold increase in [32P]Ser; NK + C1R — 10′: 5.4-fold increase in  [32P]Tyr, 2.5-fold increase in [32P]Ser.
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Figure 4: 32P incorporation into NK cell–derived Syk. 107 32P-labeled NK cells were mixed with 5 × 106 cells of the indicated target (K562 or C1R), pelleted, and incubated at 37°C for the indicated times. Syk immunoprecipitates were resolved by SDS-PAGE, transferred to membrane, and exposed to x-ray film (Syk) followed by probing with antiphosphotyrosine mAb (P-tyr blot). cpm from phosphoamino acid analysis were: NK + K562 — 5′: 2.3-fold increase in [32P]Tyr, 2-fold increase in [32P]Ser; NK + K562 — 10′: 1.5-fold increase in [32P]Tyr, 1.5-fold increase in [32P]Ser; NK + C1R — 5′: 2.4-fold increase in [32P]Tyr, 1.6-fold increase in [32P]Ser; NK + C1R — 10′: 5.4-fold increase in [32P]Tyr, 2.5-fold increase in [32P]Ser.

Mentions: Although Syk activation has not been described in tumor cells undergoing cell death, we could not formally exclude the possibility that the endogenous Syk in the above panel of targets was undergoing tyrosine phosphorylation. To ensure that the observed increase in tyrosine phosphorylation was NK cell–derived Syk, NK cell clones were labeled with [32P]orthophosphate before cellular target stimulation. Indeed, Syk immunoprecipitates showed an increase in 32P incorporation of a band at 72 kD after stimulation (Fig. 4). A phosphotyrosine blot overlays a 72-kD radiolabeled band on the autoradiogram. Phosphoaminoacid analysis of the 72-kD band illustrates that the increase in 32P incorporation is due to an increase in phosphotyrosine and phosphoserine (data not shown). These data support the conclusion that NK cell–derived Syk is tyrosine phosphorylated after stimulation with NK-sensitive tumor targets.


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)

32P incorporation into NK cell–derived Syk. 107 32P-labeled  NK cells were mixed with 5 × 106 cells of the indicated target (K562 or  C1R), pelleted, and incubated at 37°C for the indicated times. Syk immunoprecipitates were resolved by SDS-PAGE, transferred to membrane, and exposed to x-ray film (Syk) followed by probing with antiphosphotyrosine mAb (P-tyr blot). cpm from phosphoamino acid analysis  were: NK + K562 — 5′: 2.3-fold increase in [32P]Tyr, 2-fold increase in  [32P]Ser; NK + K562 — 10′: 1.5-fold increase in [32P]Tyr, 1.5-fold increase in [32P]Ser; NK + C1R — 5′: 2.4-fold increase in [32P]Tyr, 1.6-fold increase in [32P]Ser; NK + C1R — 10′: 5.4-fold increase in  [32P]Tyr, 2.5-fold increase in [32P]Ser.
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Figure 4: 32P incorporation into NK cell–derived Syk. 107 32P-labeled NK cells were mixed with 5 × 106 cells of the indicated target (K562 or C1R), pelleted, and incubated at 37°C for the indicated times. Syk immunoprecipitates were resolved by SDS-PAGE, transferred to membrane, and exposed to x-ray film (Syk) followed by probing with antiphosphotyrosine mAb (P-tyr blot). cpm from phosphoamino acid analysis were: NK + K562 — 5′: 2.3-fold increase in [32P]Tyr, 2-fold increase in [32P]Ser; NK + K562 — 10′: 1.5-fold increase in [32P]Tyr, 1.5-fold increase in [32P]Ser; NK + C1R — 5′: 2.4-fold increase in [32P]Tyr, 1.6-fold increase in [32P]Ser; NK + C1R — 10′: 5.4-fold increase in [32P]Tyr, 2.5-fold increase in [32P]Ser.
Mentions: Although Syk activation has not been described in tumor cells undergoing cell death, we could not formally exclude the possibility that the endogenous Syk in the above panel of targets was undergoing tyrosine phosphorylation. To ensure that the observed increase in tyrosine phosphorylation was NK cell–derived Syk, NK cell clones were labeled with [32P]orthophosphate before cellular target stimulation. Indeed, Syk immunoprecipitates showed an increase in 32P incorporation of a band at 72 kD after stimulation (Fig. 4). A phosphotyrosine blot overlays a 72-kD radiolabeled band on the autoradiogram. Phosphoaminoacid analysis of the 72-kD band illustrates that the increase in 32P incorporation is due to an increase in phosphotyrosine and phosphoserine (data not shown). These data support the conclusion that NK cell–derived Syk is tyrosine phosphorylated after stimulation with NK-sensitive tumor targets.

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