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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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ZAP-70 is not required for natural cytotoxicity. (A) Whole  cell lysates from normal and ZAP-70–deficient NK cells were resolved by  SDS-PAGE, transferred to membrane, and probed with anti–ZAP-70 antisera (ZAP-70) or anti-Syk mAb (Syk). (B) Normal and ZAP-70–deficient NK cells were incubated for 4 h with 51Cr-labeled 721.221 cells.
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Figure 7: ZAP-70 is not required for natural cytotoxicity. (A) Whole cell lysates from normal and ZAP-70–deficient NK cells were resolved by SDS-PAGE, transferred to membrane, and probed with anti–ZAP-70 antisera (ZAP-70) or anti-Syk mAb (Syk). (B) Normal and ZAP-70–deficient NK cells were incubated for 4 h with 51Cr-labeled 721.221 cells.

Mentions: Although the data using piceatannol are consistent with a required role for Syk family PTK in the development of natural cytotoxicity, conclusions from pharmacologic approaches must be tempered by concerns regarding potential drug effects on other second messenger molecules. To further define a role for Syk in natural cytotoxicity, we took a genetic approach to express wild-type or kinase-inactive forms of Syk family members and to evaluate the effects on natural cytotoxicity. We used the vaccinia virus expression system to overexpress wild-type or kinase-inactive forms of the two Syk family members, Syk and ZAP-70, in NK cell clones. Two different Syk mutations were used. The SykT mutant, which has recently been shown to act as a dominant-negative in mast cells (35), is truncated at residue 395 so as to lack the kinase domain but contain the SH2 domains. The kinase-inactive SykK mutant has a point mutation at residue 395 (K to R) that is within the ATP-binding region of the kinase domain. Kinase inactive ZAP-70 (ZAP-70K) also has a K to R point mutation at residue 369 within the kinase domain. After a 4 h infection with virus containing the coding sequences either for wild-type Syk family PTK, mutant Syk family PTK, or the control vector, NK cell function was assessed in a 51Cr-release assay. Overexpression of wild-type Syk with vaccinia enhanced natural killing whereas wild-type ZAP-70 overexpression did not (Fig. 6). The kinase-inactive mutants of Syk (SykT, SykK) but not ZAP-70 (ZAP-70K) acted as dominant-negatives to inhibit natural killing (Fig. 6). Although the latter observation is consistent with the notion that ZAP-70 is not required for natural cytotoxicity, one cannot exclude the possibility that the inability of ZAP-70K to inhibit killing is due to insufficient expression of the transfected gene product. Therefore, we took a genetic approach to directly analyze this issue. Specifically, we derived NK cell clones from a patient with ZAP-70 deficiency (Fig. 7 A) and found that these clones mediated wild-type levels of natural killing (Fig. 7 B) as previously reported (21, 22). Taken together, the above results suggest a required role for Syk, but not ZAP-70, in natural killing. It should be emphasized that although our data indicate that ZAP-70 activity is not necessary for natural cytotoxicity, they do not exclude the possibility that under normal circumstances ZAP-70 may subserve some signaling role.


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)

ZAP-70 is not required for natural cytotoxicity. (A) Whole  cell lysates from normal and ZAP-70–deficient NK cells were resolved by  SDS-PAGE, transferred to membrane, and probed with anti–ZAP-70 antisera (ZAP-70) or anti-Syk mAb (Syk). (B) Normal and ZAP-70–deficient NK cells were incubated for 4 h with 51Cr-labeled 721.221 cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199178&req=5

Figure 7: ZAP-70 is not required for natural cytotoxicity. (A) Whole cell lysates from normal and ZAP-70–deficient NK cells were resolved by SDS-PAGE, transferred to membrane, and probed with anti–ZAP-70 antisera (ZAP-70) or anti-Syk mAb (Syk). (B) Normal and ZAP-70–deficient NK cells were incubated for 4 h with 51Cr-labeled 721.221 cells.
Mentions: Although the data using piceatannol are consistent with a required role for Syk family PTK in the development of natural cytotoxicity, conclusions from pharmacologic approaches must be tempered by concerns regarding potential drug effects on other second messenger molecules. To further define a role for Syk in natural cytotoxicity, we took a genetic approach to express wild-type or kinase-inactive forms of Syk family members and to evaluate the effects on natural cytotoxicity. We used the vaccinia virus expression system to overexpress wild-type or kinase-inactive forms of the two Syk family members, Syk and ZAP-70, in NK cell clones. Two different Syk mutations were used. The SykT mutant, which has recently been shown to act as a dominant-negative in mast cells (35), is truncated at residue 395 so as to lack the kinase domain but contain the SH2 domains. The kinase-inactive SykK mutant has a point mutation at residue 395 (K to R) that is within the ATP-binding region of the kinase domain. Kinase inactive ZAP-70 (ZAP-70K) also has a K to R point mutation at residue 369 within the kinase domain. After a 4 h infection with virus containing the coding sequences either for wild-type Syk family PTK, mutant Syk family PTK, or the control vector, NK cell function was assessed in a 51Cr-release assay. Overexpression of wild-type Syk with vaccinia enhanced natural killing whereas wild-type ZAP-70 overexpression did not (Fig. 6). The kinase-inactive mutants of Syk (SykT, SykK) but not ZAP-70 (ZAP-70K) acted as dominant-negatives to inhibit natural killing (Fig. 6). Although the latter observation is consistent with the notion that ZAP-70 is not required for natural cytotoxicity, one cannot exclude the possibility that the inability of ZAP-70K to inhibit killing is due to insufficient expression of the transfected gene product. Therefore, we took a genetic approach to directly analyze this issue. Specifically, we derived NK cell clones from a patient with ZAP-70 deficiency (Fig. 7 A) and found that these clones mediated wild-type levels of natural killing (Fig. 7 B) as previously reported (21, 22). Taken together, the above results suggest a required role for Syk, but not ZAP-70, in natural killing. It should be emphasized that although our data indicate that ZAP-70 activity is not necessary for natural cytotoxicity, they do not exclude the possibility that under normal circumstances ZAP-70 may subserve some signaling role.

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