By
From the * Department of Immunology, and the Division of Allergy, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
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.
The NK cell is a type of lymphocyte that is able to mediate natural cytotoxicity against a variety of tumor
cells, virus-infected cells, and hematopoietic targets (1).
However, little is known regarding the mechanisms by
which these targets trigger natural cytotoxicity. In addition
to natural cytotoxicity, the NK cell can mediate antibody-dependent cell-mediated cytotoxicity (ADCC)1 through its
FcR for IgG, Fc Evidence using the PTK inhibitors herbimycin A and
genistein demonstrates that PTK activation is necessary for
both FcR-mediated and natural cytotoxicity (10). However, the available genetic evidence from Lck- and Fyn-deficient mice suggests that neither of these specific Src family
tyrosine kinases is necessary for NK cell-mediated cytotoxicity (15, 16). This raises the possibility that another Src
family PTK (e.g., Lyn) could subserve this role. Alternatively, the receptors initiating NK cell-mediated killing could use a different kind of PTK that might function in a
Src family kinase-independent manner. The Syk family
member ZAP-70, which is expressed in NK cells, is unlikely to be this PTK because ZAP-70 appears to require
activation by a Src family PTK (17); and because NK
cells from humans and mice lacking ZAP-70 can mediate normal natural cytotoxicity and ADCC (21). In contrast, Syk itself could perform this role. Syk is expressed in
NK cells (24) and evidence suggests that Syk is able to
function in the absence of Src family kinases (18, 25).
Although chimeric receptors containing the intracellular
portion of ZAP-70 require cross-linking with Src family
kinases such as Lck or Fyn to mediate cytotoxicity, chimeric receptors containing Syk can initiate killing in the absence of Src family PTK costimulation (18). In addition,
Syk, but not ZAP-70, can induce the tyrosine phosphorylation of ITAM-containing receptor subunits in an Src
family PTK-independent manner (19, 20, 25).
To test the hypothesis that the Syk tyrosine kinase is
functionally involved in natural cytotoxicity, we first stimulated NK cells with a panel of sensitive targets and then
characterized their intracellular signaling events. Although
stimulation of NK cells with sensitive targets is known to
induce increases in intracellular free Ca2+ and inositol phosphate release (26), more proximal specific signaling events
have not been elucidated. In this study, we describe a rapid
and reversible increase in the tyrosine phosphorylation of
NK cell-derived Syk after target cell stimulation. In addition, either pharmacologic inhibition of Syk kinase activity
or expression of dominant-negative, kinase-inactive Syk in
NK cells inhibits natural killing. Finally, tumor cells that
are made resistant to NK cell-mediated cytotoxicity by
MHC class I transfection no longer activate Syk. Together
these data emphasize the central contribution of the Syk tyrosine kinase to generation of natural cytotoxicity.
Cells, Chemicals, and Abs.
The P815 cell line (murine mastocytoma) was obtained from American Type Culture Collection
(Rockville, MD). The HLA class I-deficient C1R cell line
(B-LCL) and its HLA-transfected derivatives were supplied by
Peter Cresswell (Yale University, New Haven, CT). The HLA
class I-deficient cell line 721.221 (B-LCL) was provided by Peter
Parham (Stanford University, Palo Alto, CA). Human PBL were
isolated from defibrinated blood by Ficoll-Hypaque density centrifugation. Human CD16+ NK cell lines were isolated and characterized as previously described (29). All chemicals, unless otherwise noted, were obtained from Sigma Chemical Co. (St. Louis,
MO). The 3G8 hybridoma (anti-Fc Cytotoxicity Assays.
The 51Cr-release assays measuring direct
NK cell-mediated cytotoxicity were performed as previously described (29). For experiments evaluating the effects of piceatannol
(Boehringer Mannheim, Indianapolis, IN), cloned human NK
cells were pretreated with the indicated concentration of the drug
for 15 min at 37°C. Lytic units were calculated based on 20% cytotoxicity (33).
Vaccinia Viruses.
ZAP-70K cDNA was excised from pcDNA3.
KD.myc.Zap70 (provided by Lawrence E. Samelson, National
Institutes of Health, Bethesda, MD; reference 34) with BamHI/
NotI, and blunt-ended with Klenow. The blunt-ended ZAP-70K
fragment was inserted into the SmaI cloning site of the vector
pSC11 and introduced into WR strain vaccina virus via homologous recombination. Recombinant vaccinia viruses encoding
wild-type Syk, wild-type ZAP-70, SykT, and SykK as well as the
pSC-65 vector were provided by Jean-Pierre Kinet and Andrew M. Scharenberg (Harvard Medical School, Boston, MA; references 35, 36). For infections, NK cells (2 × 106 cells/ml) were
incubated in serum-free RPMI 1640 for 1 h at 37°C at a multiplicity of infection of 20. Cells were then incubated for the remainder of the indicated infection time at 106 cells/ml in RPMI
1640 supplemented with 10% bovine calf serum.
Whole Cell Lysates, Cell Stimulation, Immunoprecipitation, and Immunoblot Analysis.
For whole cell lysates, 1.5 × 106 NK cells
were lysed in buffer containing 10 mM Tris (pH 7.4), 50 mM
NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, and 1%
Triton X-100 (pH 7.4). Protein levels were determined using the
BCA Protein Assay (Pierce, Rockford, IL) and equal amounts of
protein were resolved by SDS-PAGE. For cell stimulation, NK
cells were resuspended at 108/ml and target cells were resuspended at 5 × 107/ml in RPMI 1640 supplemented with 0.5%
BSA. 100 µl of NK cells were mixed with 100 µl of target cells
(effector/target ratio of 2:1), pelleted at 5,000 rpm for 5 s, and
then incubated at 37°C for the indicated time. For anti-FcR stimulation, NK cells were incubated at 4°C for 3 min with anti-FcRIII. In contrast to natural cytotoxicity, ADCC is well defined with respect to the receptor-ligand
interaction as well as intracellular second messenger involvement (for review see reference 2). In spite of this contrast between the well-defined FcR-dependent and the
poorly defined natural cytotoxic mechanisms, both killing
mechanisms can be inhibited by MHC-recognizing killer cell inhibitory receptors (KIR). Since KIR inhibition
seems to target signaling pathways initiated by immunoreceptor tyrosine-based activation motif (ITAM)-containing
receptor complexes (e.g., Fc
RIII, TCR, and Fc
RI; references 3), the ability of both FcR-dependent and natural cytotoxicity to be inhibited by KIR suggests that there
may be shared signaling elements in the pathways used during the two alternative routes of initiating NK cell-mediated cytotoxicity.
RIII) was provided by Bice
Perussia (Thomas Jefferson University, Philadelphia, PA; reference 31) and 3G8 mAb was purified by affinity chromatography over protein A agarose. The generation and characterization of rabbit antisera specific for ZAP-70 have been previously described (32). The 4D10 mAb specific for Syk was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology
Inc. (Lake Placid, NY).
R mAb (3G8; 10 µg/ml). Washed cells were then mixed
with goat anti-mouse IgG F(ab
)2 fragments (Organon Teknika-Cappel, Durham, NC), rapidly pelleted, and incubated for 1 min
at 37°C. After stimulation, cells were lysed in buffer containing
20 mM Tris-HCl, 40 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 0.1% BSA, 1 mM Na3VO4, 1 mM PMSF, 5 µg/
ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100 (pH 7.4). Samples were centrifuged at 15,000 g for 5 min to remove insoluble material. Supernatants were subjected to immunoprecipitation with either rabbit antiserum specific for ZAP-70 bound
to protein A-Sepharose beads or with anti-Syk mAb 4D10
bound to either protein A-Sepharose or goat anti-mouse IgG
agarose beads. Immunoprecipitates were washed and bound proteins were eluted with 50 µl of SDS-sample buffer. Proteins resolved by SDS-PAGE were electrophoretically transferred to Immobilon-P membranes (Millipore, Bedford, MA).
32P-labeling and Phosphoamino Acid Analysis. NK cells were washed with phosphate-free RPMI 1640 medium supplemented with 4% bovine calf serum and 1% L-glutamine. Cells were labeled with 0.5 mCi of [32P]orthophosphate/ml for 3 h at 37°C at 1.5 × 107 cells/ml in phosphate-free medium, washed twice, and then resuspended at 108 cells/ml, and 100 µl of NK cells were used for stimulation. Target cells were also washed with phosphate-free media (not containing [32P]orthophosphate) before use for cell stimulation.
For phosphoamino acid analysis, portions of the membrane corresponding to Syk on the autoradiogram were cut and boiled in 6 N HCl for 1 h at 110°C to elute the protein. Membrane fragments were rinsed twice with water and rinses were combined with eluate. Eluate plus rinse was dried in a speed-vac and resuspended in water/ethanol/methanol (10:5:1) before spotting on thin-layer chromatography (TLC) plates. Two cycles were run in solvent ethanol/ammonium hydroxide/water (105:42:6) with phosphoamino acid standards. TLC plates were sprayed with 0.2% ninhydrin in ethanol before exposure to film.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). 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.
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.
Piceatannol Pretreatment Inhibits Natural Cytotoxicity.
Although the above data demonstrate biochemically that Syk
is modified in NK cells after target cell stimulation, a functional requirement of Syk for cytotoxicity remained to be
demonstrated. To assess whether Syk family tyrosine kinase
activity is required for natural cytotoxicity, we used the
PTK inhibitor piceatannol (38). Syk has previously been
shown to be 10-fold more sensitive to piceatannol treatment than Lyn, an Src family member, in in vitro kinase assays (38). Pretreatment of NK cell clones with piceatannol
inhibited natural cytotoxicity and FcR-initiated killing in a
concentration-dependent fashion with half-maximal inhibition at a concentration of ~6 µg/ml (Fig. 5). This 50%
inhibitory concentration is consistent with the concentration that exhibits specific inhibition of Syk in vitro kinase
activity. These results suggest that the tyrosine kinase activity of the Syk family is functionally required for natural cytotoxicity.
Syk Overexpression Enhances Natural Cytotoxicity Whereas Kinase-inactive Syk Expression Inhibits Killing.
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.
NK-resistant Targets Fail to Induce Syk Tyrosine Phosphorylation.
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.
NK cells exhibit unprimed, natural cytotoxicity against many different targets (1). Here we examine the mechanisms by which NK cells generate cytotoxicity against a panel of tumor targets. We demonstrate that the PTK Syk plays a central role in NK cell-mediated cytotoxicity.
Mechanisms of natural cytotoxicity have been difficult to
study due to the lack of defined triggering receptors. A
number of candidate receptors have been described which
can induce certain aspects of NK cell activation and/or cytotoxicity. Among these receptors are adhesion molecules
such as the 1- and
2-integrins (40), and other receptors such as CD2 (45), CD69 (46), Lag-3 (47), DNAM-1
(48), KAR (4, 49), NK-TR (58), and NKR-P1 (59-
61). Antibodies to some of these receptors can enhance the killing of FcR-bearing resistant targets ("reverse ADCC")
as well as stimulating the release of inositol phosphates and
intracellular free Ca2+. However, proving the direct involvement of such receptors in the natural killing of specific tumor targets has often been problematic. Given the
number of NK cell receptors that are candidate triggering receptors and the broad range of sensitive target cells, the
receptor-ligand interactions involved may be relatively
specific for each effector-target combination. In addition to
the mere presence or absence of triggering receptors on
NK cells or triggering ligands on target cells, the distribution of these proteins in the cell membrane may be critical. Helander et al. (44) propose the
2-integrin LFA-1 as a receptor able to trigger natural cytotoxicity by recognizing its
redistributed ligand ICAM (intracellular adhesion molecule)-2 on diseased versus normal cells. Their results suggest that when ICAM-2 is concentrated on the target cell
membrane, LFA-1 can participate in triggering cytotoxicity. Given the potential heterogeneity in triggering receptors required for natural cytotoxicity and the possibility that
ligand distribution on the target cell may be involved, we used a panel of NK-sensitive tumor targets to stimulate NK
cell clones rather than isolating receptor-ligand interactions
to examine the molecular mechanisms involved in natural
killing. Indeed, our data suggest that for this panel of targets, the Syk tyrosine kinase plays a common regulatory
role in the generation of natural cytotoxicity.
The molecular mechanisms by which Syk is activated
during natural cytotoxicity are unknown. In contrast to the
poorly defined receptor-ligand interactions involved in
natural cytotoxicity, FcR-initiated killing has a well-defined
receptor and signaling pathway. FcRIII consists of a ligand-binding
chain noncovalently associated with homo- or
heterodimers of the CD3-
and Fc
RI
chains (62).
The
and
chains contain ITAMs that are thought to
serve as substrates for Src family PTK (for reviews see references 66 and 67). Once these ITAMs are phosphorylated
by Src family PTK, Syk family PTKs can bind to the
ITAM via their SH2 domains and activate downstream
pathways. Fc
RIII can induce tyrosine phosphorylation
and activation of both Syk and ZAP-70 (24, 32, 37) but,
similar to natural cytotoxicity, expression of dominant-negative, kinase-inactive forms of Syk but not ZAP-70 inhibit FcR-initiated killing (data not shown). This suggests
that although Syk and ZAP-70 are activated after FcR
cross-linking, ZAP-70 is not required for FcR-mediated
cytotoxicity. These results are consistent with results from
ZAP-70-deficient humans and mice showing no defects in
natural cytotoxicity or ADCC (21).
Since our data suggest that Syk is a common signaling element used by FcR-initiated killing and natural cytotoxicity and since Syk family kinases are thought to dock to
ITAM-containing molecules, this raises the possibility that
ITAM-containing receptor complexes might be involved
in natural cytotoxicity. The known ITAM-containing subunits expressed in NK cells, and
, do not appear to subserve this role in initiating natural cytotoxicity since NK
cells from animals lacking these genes mediate normal natural killing (68). Killer cell activating receptors (KARs),
variants of the KIRs that lack the cytoplasmic inhibitory
motif required to bind the phosphatase SHP-1, have the
capability to initiate NK cell-mediated killing (4, 49).
Recent data suggest that KARs coprecipitate proteins with
molecular weights of ~12-16 kD (KAR-associated proteins or KARAPs) that are not
or
(71). Although the
molecular identities of these molecules are unknown, two-dimensional gel analysis suggests that, similar to
and
,
they may be expressed as disulfide-linked dimers. Expression of KARAPs appears to correlate with the ability of
KARs to activate granule release, since cross-linking KAR
on NK cells, which express KARAP, can trigger cytotoxicity while cross-linking KAR-transfected rat basophilic leukemia cells, which do not express detectable KARAP, does
not induce granule release (71). Molecular cloning of the
KARAP protein will be needed to determine if these are
indeed additional ITAM-containing subunits that are
shared by other triggering-receptor complexes.
Our data clearly suggest that Syk and ZAP-70 are not
functionally redundant in the generation of natural cytotoxicity. This observation is consistent with reports demonstrating different activation requirements for each of the
Syk family members. In vitro studies using peptides containing phosphorylated ITAMs from the FcRI
chain
suggest that these peptides can activate Syk kinase activity
(72, 73), whereas ZAP-70 kinase activity is only activated
upon binding a dimeric form of phosphorylated
(74)
rather than peptide fragments (75, 76). In vivo studies in
COS cells suggest that ZAP-70 is only phosphorylated
upon the addition of Lck or Fyn (17), whereas Syk is phosphorylated when transfected alone (77, 78). The intrinsic
enzymatic activities of Syk and ZAP-70 also differ when
expressed in COS cells (79). Syk autophosphorylation and
catalytic activity toward the exogenous substrate erythrocyte band 3 was ~100-fold greater than that of ZAP-70.
Further studies in COS cells expressing chimeric receptors which cytoplasmically express Fc
RI
chain coexpressed
with Syk or ZAP-70 showed that the chimeric
plus Syk
led to
phosphorylation and Syk kinase activity while
plus ZAP-70 induced neither
phosphorylation nor ZAP-70 activation (20).
The final outcome of an NK cell interacting with a target cell is determined by the balance of positive and negative signals transmitted. Positive signals generated during
natural cytotoxicity include activation of PTKs and increases in inositol phosphate and intracellular free Ca2+ release. Negative signals are derived, at least in part, from the
protein-tyrosine phosphatase (PTPase) SHP-1 that is associated with MHC-recognizing KIR (5, 8, 80). Although it appears the overall balance of positive and negative signals generated determines the outcome of an NK
cell interacting with a target cell, our data suggest that the
two pathways may directly interact. KIR may preempt the
initiation of a positive signal by acting on the activating receptor complex or its most proximal signaling molecules.
Since Syk activation is central to the generation of both
natural cytotoxicity and ADCC, SHP-1-mediated dephosphorylation of Syk could inhibit cellular activation. Alternatively, SHP-1 may act by inactivating a kinase that may
phosphorylate and activate Syk. Recent data suggest that
Syk may be activated by an Src kinase-initiated activation
loop in which, once a small amount of Syk is phosphorylated, it can activate other Syk molecules to allow rapid Syk
activation (36). Another potential SHP-1 target is the
ITAM-containing receptor protein that upon phosphorylation docks Syk family molecules to the receptor complex.
SHP-1 targeting any of these activation steps could inhibit
the Syk tyrosine kinase by either preventing the association
of Syk with the receptor complex or by preventing its full
activation. Valiante et al. (83) have suggested that KIR-associated SHP-1 inhibits NK cell-mediated natural killing by
dephosphorylating such downstream targets as the phospholipase C (PLC)--adaptor protein pp36, thereby preventing PLC-
association and activation. Our results here,
together with previous data from our lab (4,5), suggest that
inhibition may occur at a more proximal step in activation.
If inhibition of Syk activation, a proximal step in the generation of natural cytotoxicity and ADCC, is the target for
KIR inhibition, then this supports a role for Syk as a common second messenger in use during many forms of cytotoxicity.
Address correspondence to Dr. Paul J. Leibson, Department of Immunology, Mayo Clinic and Foundation, Rochester, MN 55905. Phone: 507-284-4563; FAX: 507-284-1637; E-mail: leibson.paul{at}mayo.edu
Received for publication 7 August 1997 and in revised form 9 October 1997.
This research was supported by the Mayo Foundation and by grant CA-47752 from the National Institutes of Health.The authors wish to thank Jean-Pierre Kinet and Andrew M. Scharenberg for generously providing the pSC-65, Syk, ZAP-70, SykT, and SykK recombinant vaccinia viruses, and Theresa Lee for her skillful assistance with the preparation of the manuscript.
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