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Mentions: Although PTK activation is an early and requisite event in the generation of natural cytotoxicity (10, 11), genetic evidence suggests that the PTKs Lck, Fyn, and ZAP-70 are not required (15, 16, 21–23). These observations prompted us to evaluate whether the tyrosine kinase Syk is important for natural cytotoxicity. We first determined whether Syk was tyrosine phosphorylated after stimulation of NK cells with sensitive targets since an increase in Syk tyrosine phosphorylation correlates with increased Syk activation. Human NK cell clones were incubated either with the MHC class I–deficient B lymphoblastoid targets C1R or 721.221 or with the prototypic human NK cell target K562. After various times of incubation, cells were lysed and Syk was immunoprecipitated. We observed an increase in Syk tyrosine phosphorylation after NK cells were stimulated with each of the panel of targets (Fig. 1, A–C). This increase in Syk tyrosine phosphorylation was a relatively early event, peaking at 5 min and declining to baseline by 60 min (Fig. 2). Since both Syk family members Syk and ZAP-70 are expressed in NK cells and activated downstream of the FcR (24, 32, 37), we questioned whether NK cell–derived ZAP-70 was also tyrosine phosphorylated after incubation with sensitive targets. ZAP-70 immunoprecipitates from target cell–stimulated lysates revealed no increase in the tyrosine phosphorylation of ZAP-70 after target cell contact (Fig. 1, A–C). This was not a defect in the ability of ZAP-70 to be phosphorylated in these NK cells since cross-linking with FcR-specific antibodies on the same cells results in an increase in ZAP-70 tyrosine phosphorylation (Fig. 1, A–C). Thus, it appears that Syk and not ZAP-70 is activated when this panel of targets is used to stimulate NK cells. Consistent with the observations that both Syk and ZAP-70 are tyrosine phosphorylated after FcR ligation (24, 32, 37), stimulation of NK cells with the 3G8 hybridoma, which expresses membrane-bound antibody to the FcR, resulted in an increase in both Syk and ZAP-70 tyrosine phosphorylation (Fig. 3). These data suggest that while FcR cross-linking can stimulate the tyrosine phosphorylation of Syk and ZAP-70, stimulation of NK cells with this panel of sensitive targets results in the phosphorylation of Syk without detectable ZAP-70 phosphorylation.
Functional Role for Syk Tyrosine Kinase in Natural Killer Cell–mediated Natural Cytotoxicity
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.
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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