From the
Ligation of the Fc
Exposure to foreign antigens elicits an antibody response from
the immune system. The subsequent interaction of Fc receptor-bearing
effector cells with antibodies complexed to soluble or cell-bound
antigens initiates a variety of effector functions. Natural killer
(NK)
Elegant studies by
several groups have demonstrated that ligation of chimeric receptors
containing the intracellular portions of either the
We focused
our initial examinations of the PTKs involved in Fc
Immunodetection with rabbit antisera specific for ZAP-70,
PLC-
Biochemical studies have yielded valuable insights into the
signaling machinery triggered by the Fc
The role of lck in signal
transduction has been studied extensively in T cells. The seminal
observations that lck associates with the CD4 and CD8 coreceptors in T
cells and that cross-linking of these coreceptors increases lck kinase
activity suggest a critical function for lck during antigen-induced T
cell
activation(43, 44, 45, 46, 47) .
Aside from its interaction with CD4 and CD8, lck appears to be
important as well in CD4/CD8-independent TCR signaling as emphasized by
studies with genetic mutants of T cell lines. lck-deficient variants of
the human Jurkat and the mouse CTLL-2 T cell lines exhibit impaired TCR
signaling and effector functions(29, 48) . Conversely,
overexpression of an activated mutant of lck enhances TCR-mediated
signaling in a T cell hybridoma (49). In contrast to T cells, the role
of lck in Fc
Since
several different members of the src family PTKs are expressed in NK
cells(50) , we tested whether these related PTKs exhibited any
specificity in mediating Fc
Since the role of syk family PTKs in Fc
Our
finding that lck has a greater effect on the Fc
Fc
In summary, this study suggests a
regulatory role for lck in the Fc
We thank Roger Perlmutter, Bart Sefton, and Sally
Parsons for generous gifts of the src family cDNAs. We are also
grateful to Hans Schreiber for providing the pSC11 vector, Augusto
Ochoa for the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
R on natural killer (NK) cells results in
the tyrosine phosphorylation of multiple substrates critical for
intracellular signaling and activation of NK cell effector functions.
However, it remains unclear which nonreceptor protein-tyrosine kinases
(PTK) participate in this process. In this report we demonstrate that
Fc
R ligation induced the tyrosine phosphorylation and increased
the catalytic activities of both syk family PTKs, ZAP-70, and syk. The
phosphorylation of ZAP-70 and syk was enhanced markedly by
overexpression of wild-type lck but not by a kinase-inactive mutant,
suggesting that early Fc
R-initiated activation of lck results in
the subsequent regulation of syk family PTKs. The regulatory interplay
between src and syk family PTKs was emphasized further by the
observation that lck overexpression enhanced the association of ZAP-70
with the
chain of the Fc
R complex. Additional analyses
indicated that lck induced the subsequent tyrosine phosphorylation of
phospholipase C (PLC)-
2. Interestingly, the regulatory effects of
lck on ZAP-70, syk, and PLC-
2 could not be replaced by
overexpression of either fyn or src, demonstrating a selective role for
lck in effectively coupling Fc
R stimulation to critical downstream
signaling events. Taken together, our results suggest not only that
Fc
R stimulation on NK cells is coupled to the intracellular
activation of both ZAP-70 and syk, but that the src family member, lck,
can selectively regulate this tyrosine kinase cascade.
(
)cells represent a distinct subpopulation
of lymphocytes which express the IgG Fc receptor type IIIA, hereafter
referred to as Fc
R(1) . The Fc
R on human NK cells is a
multimeric receptor complex consisting of the ligand-binding
subunit (i.e. CD16), which associates noncovalently with
dimers of
and
chains(2) . Although none of the
components of the receptor complex possesses intrinsic kinase activity,
stimulation of the Fc
R rapidly activates a protein-tyrosine kinase
(PTK) signaling pathway that results in the tyrosine phosphorylation of
substrates critical for cellular activation, including the
chain
and phospholipase C (PLC)-
isoforms(3, 4, 5, 6, 7) . This
Fc
R-initiated PTK signaling pathway appears to be requisite for
the activation of NK cell-mediated cytotoxicity and lymphokine
production(8, 9, 10) .
or
chains is sufficient to activate intracellular PTKs as well as mediate
downstream cytolytic function and lymphokine production (11-14).
Additional studies have identified a conserved motif in the
and
chains, with the consensus amino acid sequence
YXXL-X
-YXXL, which is
sufficient to mediate intracellular signaling(15, 16) .
src family members are candidates for the Fc
R-associated PTKs.
Recent reports have shown that lck, a member of the src family, is
detectable in anti-Fc
R immunoprecipitates and that ligation of
Fc
R increases the in vitro catalytic activity of
lck(10, 17, 18) . Nonetheless, the precise
regulatory function of lck during Fc
R-initiated signaling remains
unclear. The syk family PTKs represent an additional class of
cytoplasmic PTKs that have been implicated in lymphoid cell signal
transduction. T cell antigen receptor (TCR) ligation has been shown to
induce the tyrosine phosphorylation of ZAP-70 and its association with
the
chains of activated TCR complexes(19, 20) .
Furthermore, a deficiency in ZAP-70 expression severely impairs
TCR-mediated signaling(21, 22) . Likewise, stimulation
of the B cell antigen receptor (BCR) results in the tyrosine
phosphorylation of the receptor-associated syk(23, 24) .
In contrast to T and B cells, the function of syk family PTKs in NK
cell activation is less clear. Ligation of the Fc
R on NK cells can
induce the association of a 70-kDa phosphotyrosyl protein with the
receptor complex(25) , but the precise roles of ZAP-70 and/or
syk during Fc
R signal transduction and the nature of their
potential interaction with src family PTKs are unknown.
R signaling on
lck and its role in coupling Fc
R stimulation to subsequent
tyrosine phosphorylation events. Using the vaccinia virus expression
system, we overexpressed wild-type lck in cloned human NK cells.
Overexpression of wild-type active lck, but not a kinase-deficient
mutant of this PTK, markedly enhanced the Fc
R-induced tyrosine
phosphorylation of ZAP-70, syk, and PLC-
2. Furthermore, only lck
effectively coupled the Fc
R to downstream PTKs, as neither fyn nor
src could substitute for lck. Taken together, our data strongly
implicate a role for the src family PTK, lck, in the Fc
R-initiated
regulation of ZAP-70, syk, and PLC-
.
Cell Lines
Human CD16 NK cell lines were isolated and passaged as described
previously(26) . The cell surface phenotype of these NK cell
lines was monitored by flow cytometry. All NK cell lines used in these
studies were >90% CD16
. In addition, all
CD16
NK cells expressed the following additional
phenotypic markers: CD56
, CD11b
,
CD2
, and HLA-DR
.
Chemical Reagents and Antibodies
All
chemicals and drugs, unless otherwise noted, were obtained from Sigma.
Fluorescein- and phycoerythrin-conjugated monoclonal antibodies (mAb)
were obtained from Becton-Dickinson Monoclonal Center (Mountain View,
CA). The anti-FcR mAb (3G8) has been described
previously(27) . Immunoprecipitating ZAP-70 and syk rabbit
antisera and immunoblotting anti-phosphotyrosine mAb 4G10 were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Immunoblotting ZAP-70 and syk antisera were raised in rabbits injected
with keyhole limpet hemocyanin-conjugated synthetic peptides (ZAP-70
residues 326-341; 28 C-terminal amino acids of porcine
p72
)(20, 28) . The generation and
characterization of the lck and PLC-
2 rabbit antisera have been
described previously(7, 29) . The
antiserum was
generously provided by Augusta Ochoa (National Cancer Institute).
Vaccinia Viruses
Recombinant vaccinia
viruses encoding wild-type or mutant src family PTK were generated
essentially as described(30) . Blunt ended cDNA fragments were
obtained from the following sources. A a StuI fragment of
c-lck was excised from the plasmid NT18 (31) and a EcoRV-SmaI fragment of c-fyn was excised
from the plasmid pMTFR(32) , both of which were generously
provided by Roger Perlmutter (University of Washington, Seattle). A StuI fragment of lck with a lysine to arginine
mutation at position 273 was excised from a plasmid generously provided
by Bart Sefton (Salk Institute, San Diego). A HindIII fragment
of c-src was obtained from the plasmid pM5H (33) kindly
provided by Sarah Parsons (University of Virginia, Charlottesville).
These blunt ended cDNAs were inserted into the SmaI cloning
site of the vector pSC11 (34) and introduced into WR strain
vaccinia by homologous recombination. The recombinant vaccinia viruses
were characterized by infection of CV-1 cells and subsequent detection
by immunoblotting with antisera specific for the different src family
PTKs. In vitro autophosphorylation assays were also performed
on immunoprecipitated src family PTKs to confirm their catalytic
activities. Viruses were propagated in HeLa cell cultures and released
by lysing infected HeLa cells with a probe sonicator. The lysate was
then layered over a cushion of 36% sucrose solution in 10 mM Tris-HCl, pH 9.0, and centrifuged in a Beckman SW 28 rotor (13,500
rpm, 2 h) to purify the virus. Viruses were subsequently titered on
confluent BSC-1 monolayers using the method described
previously(35) . NK cells (2 10
cells/ml in
serum-free RPMI) were infected for 1 h at 37 °C at a multiplicity
of infection of 20. Cells were then incubated for an additional
3-5 h at 1
10
cells/ml in RPMI with 10%
bovine calf serum. Infected NK cells were washed twice and resuspended
in RPMI with 0.5% bovine serum albumin for stimulation.
Immunoblotting
100-µl aliquots of NK
cells (2 10
cells/sample) were incubated at 4
°C for 3 min with 10 µl of anti-Fc
R mAb (3G8) (final
concentration, 100 µg of goat F(ab`)2 fragment anti-mouse IgG
(Organon Teknika Corp., West Chester, PA) was then added to the cell
suspension, mixed, and briefly pelleted. After incubating at 37 °C
for the indicated time, pelleted cells were lysed with buffer
containing 10 mM Tris-HCl, 50 mM NaCl, 5 mM
EDTA, 50 mM NaF, 30 mM
Na
P
O
, 500 µM Na
VO
, 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton
X-100, pH 7.4. Insoluble material was removed by centrifugation at
15,000
g for 10 min, and detergent-soluble proteins
resolved by 8.5% SDS-polyacrylamide gel electrophoresis (PAGE).
Resolved proteins were electrophoretically transferred to Immobilon-P
membranes (Millipore, Bedford, MA), and immunoblotting with the
anti-phosphotyrosine mAb 4G10 was performed as described previously
(7).
2, and lck were performed as described previously(7) .
Briefly, membranes containing resolved proteins were blocked overnight
in Tris-buffered saline containing 2% milk and 0.2% polyoxyethylene
sorbitan monolaurate (Tween 20) and incubated for 1 h with the
antiserum diluted in Tris-buffered saline containing 2% bovine serum
albumin, 0.2% Tween 20, and 0.05% NaN
. After three washes
with 0.2% Tween 20 in Tris-buffered saline, immunoreactive proteins
were detected with protein A-horseradish peroxidase and the ECL
detection system from Amersham Corp.
Immunoprecipitations
200-µl aliquots
of NK cells (2 10
/sample) were incubated at 4
°C for 3 min with 10 µl of anti-Fc
R mAb (3G8) (final
concentration, 10 µg/ml). The cells were pelleted gently (700
g, 30 s, 4 °C) and resuspended in 200 µg of
goat F(ab`)
fragment anti-mouse IgG. After incubating at 37
°C for the indicated time, reactions were terminated with 1 ml of
ice-cold lysis buffer containing 20 mM Tris-HCl, 40 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM
Na
P
O
, 0.1% bovine serum albumin,
500 µM Na
VO
, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1% Triton X-100, pH 7.4. After 10 min at 4 °C, the
samples were centrifuged (15,000
g, 10 min) to remove
nuclear and cellular debris. Postnuclear supernatants were
immunoprecipitated for 1-2 h at 4 °C with rabbit antisera
bound to protein A-Sepharose beads. The immunoprecipitates were washed
three times and bound proteins eluted with 50 µl of SDS-sample
buffer and resolved by SDS-PAGE. Anti-phosphotyrosine immunoblotting
was performed as described above.
In Vitro Kinase Assay
Anti- and
anti-syk immune complex kinase reactions were performed using a
modification of the method described previously(36) . Briefly,
cell stimulation was terminated in a Brij 96 lysis buffer (1% Brij, 25
mM Tris, pH 7.6, 150 mM NaCl, 1 mM Na
VO
, 5 mM EDTA, and 10 µg/ml
aprotinin and leupeptin). Immunoprecipitates were washed two times, and
the beads were incubated at 25 °C for 5 min in 20 mM Tris,
pH 7.6, 10 mM MnCl
, 1 µM ATP, 0.25
µg of cfb3 (cytoplasmic fragment of band 3; generously provided by
Lawrence E. Samelson, NIH), 10 µCi of
[
-
P]ATP. The radiolabeled 43-kDa cfb3 was
quantitated after scanning the membrane using the Radioanalytic Imaging
System (model 4000, AMBIS, Inc., San Diego).
Vaccinia Virus Infection of Human NK
Cells
Studies on NK cells have been hampered by the lack of
a suitable methodology to genetically manipulate these cells. We report
here the use of the vaccinia virus expression systems to overexpress
efficiently src family PTKs in this cell type. Vaccinia viruses
encoding wild-type src, fyn, lck, and a
kinase-inactive lck mutant (lysine to arginine
mutation at position 273) were generated by homologous recombination.
The absence of kinase activity in the lck
mutant was verified by infection of CV-1 cells with the different
viruses followed by a lck-specific immune complex autophosphorylation
assay (data not shown). After a 4-h infection of cloned NK cells with
recombinant viruses encoding either wild-type or kinase-inactive lck, high levels of lck expression were detected by
immunoblotting with a lck-specific antiserum (Fig. 1). Likewise,
infection of NK cells with recombinant viruses encoding src or fyn resulted in the efficient overexpression of catalytically
active PTKs (data not shown). Immunoblot analyses in multiple
experiments revealed that infection of NK cells with the lck-
and fyn-encoding viruses resulted in approximately
2-5-fold increases in both lck and fyn protein relative to
endogenous levels. Furthermore, infection with either the lck-encoding or fyn-encoding viruses resulted in
2-3-fold increases in the total catalytic activity of anti-lck
+ anti-fyn immunoprecipitates for their shared substrate, enolase
(data not shown). Whereas there was no detectable src in uninfected NK
cells, infection with the src-encoding virus resulted in high
levels of src expression (data not shown). The secretory and cytotoxic
functions of NK cells remained intact during the 4-6-h infection
with the vaccinia viruses.
Figure 1:
Overexpression of lck in NK cells.
Cloned NK cells (5 10
/sample) were left uninfected
for 4 h with either the control nonrecombinant WR strain vaccinia
virus, the vaccinia virus encoding wild-type lck, or the
vaccinia encoding the mutant kinase-inactive lck
. Detergent-soluble proteins were resolved
by SDS-PAGE, transferred to Immobilon-P membrane, and probed with a
lck-specific antiserum.
lck Participates in the Fc
lck can physically
associate with the FcR-initiated Tyrosine
Phosphorylation of Multiple Proteins
R complex and exhibits enhanced in vitro catalytic activity after Fc
R ligation in NK
cells(10, 17, 18) . Furthermore, we demonstrated
in NK clones that Fc
R ligation induced a transient 2-3-fold
increase in lck-specific in vitro kinase activity, whereas no
change in fyn-specific activity was detected (data not shown). We next
examined the effects of lck overexpression on Fc
R-mediated signal
transduction. As shown in Fig. 2, overexpression of lck in
unstimulated NK cells led to the increased tyrosine phosphorylation of
several intracellular proteins (fifth lane). Interestingly,
the immunoreactive proteins migrating at molecular masses of
approximately 150, 120, 116, 85, and 75 kDa displayed electrophoretic
mobilities identical to those induced by stimulation of uninfected NK
cells with anti-Fc
R mAb (second and fifth
lanes). Fc
R ligation of lck-overexpressing NK cells led to
further increases in the tyrosine phosphorylation levels of these
substrates (sixth lane). Moreover, infection of NK cells with
either the control wild-type WR strain vaccinia (third and fourth lanes) or the recombinant virus expressing the
kinase-inactive lck (seventh and eighth
lanes) did not alter the Fc
R-induced tyrosine phosphorylation
events. We next tested whether other members of the src family PTKs can
induce the same effects as lck. In contrast to the dramatic effects
observed with lck, overexpression of fyn had only minimal effects on
the tyrosine phosphorylation levels of both resting and
Fc
R-stimulated NK cells (Fig. 3, seventh and eighth lanes). Furthermore, overexpression of src had no
detectable effects on the Fc
R-induced tyrosine phosphorylation of
proteins (Fig. 3, ninth and tenth lanes). In Fig. 3, both fyn (seventh and eighth lanes) and
src (ninth and tenth lanes) are detectable as
phosphotyrosyl proteins migrating at molecular mass of 60 kDa. These
observations are consistent with a specific role for lck in
Fc
R-initiated tyrosine phosphorylation events.
Figure 2:
lck-induced tyrosine phosphorylation in NK
cells. NK cells (2 10
/sample) were either
uninfected or infected as described in Fig. 1. Cells were subsequently
left unstimulated (-) or stimulated for 1 min with cross-linked
anti-Fc
R mAb 3G8 (+). Detergent-soluble proteins were
resolved by SDS-PAGE, transferred to Immobilon-P membrane, and
sequentially probed with the anti-phosphotyrosine mAb 4G10 (upper
panel) and with the lck antiserum (lower
panel).
Figure 3:
Effects of src family PTK overexpression
on tyrosine phosphorylation in NK cells. NK cells (2
10
/sample) were either uninfected or infected with control
WR, lck-, fyn-, or src-encoding vaccinia
virus. Fc
R stimulation and phosphotyrosine detection of cellular
lysates were performed as described in Fig.
2.
Fc
We next investigated whether members of the syk family
PTK are involved in FcR Ligation Induces the Tyrosine
Phosphorylation and Increases the Catalytic Activities of ZAP-70 and
syk
R signaling. NK cells were stimulated with
cross-linked anti-Fc
R mAb, and then either ZAP-70 or syk was
immunoprecipitated with its respective antiserum. Precipitated proteins
were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and
blotted with the anti-phosphotyrosine mAb, 4G10. Stimulation of
Fc
R rapidly elevated the phosphotyrosine levels of both ZAP-70 (Fig. 4A) and syk (Fig. 4B). Both
phosphorylation events exhibited similar kinetics, with phosphorylation
peaking at 1 min and returning to basal level by 30 min. Although the
21-23-kDa tyrosine-phosphorylated isoforms of
associated
with ZAP-70 after FcR ligation or after pervanadate-induced(37, 38) stimulation (Fig. 5), similar associations with syk
were not detected (data not shown). These results are consistent with
an earlier report of the Fc
R complex in activated NK cells
associating with a 70-kDa phosphotyrosyl protein that displayed a
peptide map similar to that of ZAP-70(25) .
Figure 4:
Kinetics of
FcR-induced tyrosine phosphorylation of ZAP-70 and syk. NK cells
(2
10
/sample) were stimulated with cross-linked
anti-Fc
R mAb 3G8 for the indicated time (minutes). ZAP-70 (panel A) or syk (panel B) immunoprecipitates were
resolved by SDS-PAGE, transferred to Immobilon-P membrane, and probed
with the anti-phosphotyrosine mAb 4G10.
Figure 5:
Kinetics of FcR-induced association
of phospho-
with ZAP-70. NK cells (2
10
/sample) were stimulated for the indicated time (min)
with either cross-linked anti-Fc
R mAb 3G8 or pervanadate (PV). ZAP-70 immunoprecipitates were resolved by SDS-PAGE
(12.5% gel), transferred to Immobilon-P membrane, and probed with the
anti-phosphotyrosine mAb 4G10.
We extended this
analysis to determine whether the FcR-induced modifications of
ZAP-70 and syk were associated with any changes in their catalytic
activity. Recent reports have demonstrated that a peptide fragment of
the human erythrocyte band 3 (i.e. cfb3) is an in vitro substrate for both ZAP-70 and syk(36, 39) . Using
this exogenous substrate in an in vitro kinase assay, we
demonstrated that FcR ligation induced rapid and kinetically similar
increases in the catalytic activities of ZAP-70 and syk, with maximal
increases by 1 min and returns toward base line by 30 min (Fig. 6). The pervanadate-induced(37, 38) tyrosine-phosphorylated forms of ZAP-70 and syk also had
increased in vitro catalytic activity (Fig. 6).
Figure 6:
FcR-induced increases in the in
vitro catalytic activities of both ZAP-70 and syk. Panel
A, NK cells (1
10
/sample) were stimulated for
the indicated times (min) with either cross-linked anti-Fc
R mAb
3G8 or pervanadate (PV). ZAP-70 or syk immunoprecipitates were
incubated in an in vitro kinase assay that included the 43-kDa
exogenous substrate, cfb3 (marker in right margin). Panel B, radiolabeled cfb3 was quantitated after scanning the
membrane using an AMBIS 4000 radioanalytic imaging
system.
Effects of lck Overexpression on ZAP-70 and
syk
The data presented thus far implicate both src and syk
family PTKs in FcR signaling. However, the precise regulatory role
of lck in the Fc
R-initiated PTK pathway, including its potential
interaction with syk family PTKs, is not known. To evaluate these
questions, NK cells were first infected with either control WR vaccinia
virus or the recombinant lck-encoding vaccinia virus and then
stimulated with cross-linked anti-Fc
R mAb (Fig. 7). Similar
to our previous observation with uninfected NK cells (Fig. 4),
cross-linking of the Fc
R on control WR-infected cells induces the
tyrosine phosphorylation of ZAP-70 (first and second
lanes) and syk (fifth and eighth lanes).
Significantly, overexpression of lck markedly enhanced the
Fc
R-induced tyrosine phosphorylation of both ZAP-70 (third and fourth lanes) and syk (seventh and eighth lanes). When the Fc
R-induced tyrosine
phosphorylation levels of ZAP-70 and syk in lck-overexpressing cells
were compared with their respective counterparts in control cells, the
enhancement was consistently greater for ZAP-70 (approximately 3-fold)
than for syk (approximately 1.5-fold) (Fig. 7). This quantitative
difference in the augmentation of the phosphorylation of ZAP-70 and syk
by lck was observed reproducibly in three separate experiments. These
effects caused by lck overexpression required an active kinase as they
were not seen with the kinase-inactive mutant (data not shown).
Furthermore, neither fyn nor src overexpression enhanced the
Fc
R-induced tyrosine phosphorylation of ZAP-70 and syk (Fig. 8). Taken together, these results suggest that lck can
selectively regulate ZAP-70 and syk during Fc
R signaling. In
addition, the quantitative difference in lck's effect on the two
related members of the syk family may reflect a difference in their
requirement for src family PTKs.
Figure 7:
lck-mediated tyrosine phosphorylation of
ZAP-70 and syk. NK cells (2 10
/sample) were
infected with either the control WR or the lck-encoding
vaccinia virus. Cells were subsequently left unstimulated (-) or
stimulated for 1 min with cross-linked anti-Fc
R mAb 3G8 (+).
ZAP-70 (first four lanes) or syk (fifth through eighth lanes) immunoprecipitates were resolved by SDS-PAGE,
transferred to Immobilon-P membrane, and probed with the
anti-phosphotyrosine mAb 4G10.
Figure 8:
Effects of src family PTK overexpression
on ZAP-70 and syk tyrosine phosphorylation. NK cells (2
10
/sample) infected with either the control WR, lck-, fyn-, or src-encoding vaccinia virus
were stimulated (+), or not (-), for 1 min with cross-linked
anti-Fc
R mAb 3G8. Phosphotyrosine detection of ZAP-70 (upper
panel) or syk (lower panel) immunoprecipitates was
performed as described in Fig. 7.
We next investigated whether
lck-mediated events regulate the association of ZAP-70 with the
FcR complex. NK cells overexpressing lck were stimulated with
cross-linked anti-Fc
R mAb, lysed in a buffer containing 1% Triton
X-100, and the
chain immunoprecipitated. Precipitated proteins
were resolved by SDS-PAGE and analyzed by immunoblotting. Detection
with the anti-phosphotyrosine mAb revealed a phosphotyrosyl-containing
protein migrating at approximately 70 kDa which associated with the
chain after Fc
R stimulation in NK cells overexpressing lck (Fig. 9, upper panel). ZAP-70 was identified among these
-associated proteins by reblotting the membrane with
ZAP-70-specific antiserum (Fig. 9, lower panel). In
parallel analyses, we were unable to detect syk among these
-associated proteins, but we cannot exclude the possibility that
this reflects decreased sensitivity of the syk-specific antiserum used
for detection. Overexpression of src, fyn (Fig. 9), or the
kinase-inactive lck (data not shown) did not have any effect on the
association of phosphotyrosyl proteins with
. Thus, the result in Fig. 9suggests that lck can function to recruit ZAP-70 to the
Fc
R complex and is consistent with the notion that lck couples the
Fc
R complex to syk family PTK activation in NK cells.
Figure 9:
lck-induced association of a
phosphotyrosyl protein with . NK cells (2
10
/sample) were infected and stimulated as described in
Fig. 6. Detergent-soluble proteins were immunoprecipitated with
anti-
antiserum, resolved by SDS-PAGE, and transferred to
Immobilon-P membrane. Phosphotyrosine-containing proteins (upper
panel) and ZAP-70 (lower panel) were detected with mAb
4G10 and anti-ZAP70 antiserum, respectively.
lck Overexpression Augments Fc
Our group and
others(5, 6, 7, 8, 9, 10) have demonstrated that tyrosine phosphorylation events,
including those involving the PLC-R-induced Tyrosine
Phosphorylation of PLC-
2
isoforms, are essential for the
initiation of Fc
R-mediated NK cell cytotoxicity. However, it
remains unclear what role src family PTK, in particular lck, may play
in mediating the activation of PLC-
isoforms. We addressed this
question by examining the effect of lck overexpression on
Fc
R-induced tyrosine phosphorylation of PLC-
2 in NK cells.
Similar to that seen previously in uninfected NK cells(7) ,
stimulation with cross-linked Fc
R induced the tyrosine
phosphorylation of PLC-
2 in either uninfected or control WR
vaccinia virus-infected NK cells (Fig. 10, first four
lanes). Upon overexpression of lck, the Fc
R-induced tyrosine
phosphorylation of PLC-
2 was enhanced dramatically (fifth and sixth lanes). In contrast, overexpression of the
kinase-inactive lck did not produce the same effect (seventh and eighth lanes). We extended our analysis to test
whether other src family PTKs may function in the same manner as lck in
regulating PLC-
2 phosphorylation. Neither fyn nor src
overexpression had the same effect as lck (data not shown). Thus, our
results implicate a possible role for lck in the regulation of
PLC-
isoforms during Fc
R-initiated activation of NK cells.
Figure 10:
lck-mediated tyrosine phosphorylation of
PLC-2. NK cells (2
10
/sample) were either
uninfected or infected with the vaccinia viruses as described in Fig.
1. Cells were subsequently stimulated (+) or not (-) for 1
min with cross-linked anti-Fc
R mAb 3G8. PLC-
2
immunoprecipitates were resolved by SDS-PAGE and transferred to
Immobilon-P membrane. The membrane was sequentially probed with the
anti-phosphotyrosine mAb 4G10 (upper panel) and PLC-
2
antiserum (lower panel).
R complex in human NK cells
(40-42). Nonetheless, although the use of molecular techniques
has greatly expanded our understanding of signal transduction in other
lymphoid cell types, the study of NK cell signaling has been restricted
by the lack of a suitable system to manipulate this cell population
genetically. We report here the successful use of the vaccinia virus
expression system to investigate the role of lck in mediating Fc
R
signal transduction. Specifically, our analyses strongly suggest that
lck can selectively regulate downstream syk family PTKs and PLC-
2
during the course of Fc
R activation.
R-initiated signal transduction in NK cells has not
been as well characterized. Although recent reports have demonstrated
the physical association of lck with the Fc
R complex in NK cells
and an induction in the catalytic activity of
lck(10, 17, 18) , the functional significance of
these observations remains to be elucidated. Thus, we conducted a
detailed examination of the involvement of lck in Fc
R-mediated
signaling in NK cells. Our preliminary characterization demonstrated
that lck overexpression led to the tyrosine phosphorylation of select
substrates, and these were enhanced further by Fc
R cross-linking.
Interestingly, most of these substrates corresponded to those that were
tyrosine-phosphorylated after Fc
R stimulation of normal NK cells,
suggesting that lck is functionally linked to the Fc
R.
R signaling. Our evaluation showed that
lck overexpression, but not that of fyn or src, resulted in the marked
elevation of tyrosine phosphorylations in NK cells. Minimal detectable
effects on the total pool of detergent-soluble phosphotyrosyl proteins
were induced by fyn overexpression, and none was detected with src
overexpression. Further examination of specific phosphorylation events,
including that of ZAP-70, syk, and PLC-
2, indicated that neither
fyn nor src could induce the same effects as lck. These observations
underline the specificity of lck in coupling the Fc
R to the PTK
signaling pathway. A recent study by Salcedo et al.(18) shows that lck, but not fyn, yes, or src, exhibits a
Nonidet P-40 detergent-stable association with both the
and
chains of the Fc
R complex. Thus, our observation that only lck
effectively coupled the Fc
R to the tyrosine phosphorylation of
downstream substrates in NK cells extends the data obtained from
coprecipitation analysis by Salcedo et al.(18) to a
functional level. Moreover, in an avian B cell system, the lack of lyn
can be replaced by lck and fyn, but not by src, demonstrating a less
restrictive specificity for src family PTKs in that experimental
system(51) .
R
signaling in NK cells has not been well characterized, we examined
whether this family of PTKs is involved. We demonstrated formally that
both PTKs of the syk family, ZAP-70 and syk, were
tyrosine-phosphorylated and activated after Fc
R stimulation.
Subsequently, we tested to see if lck acts proximal to the syk family
PTKs in Fc
R signaling. The observation that overexpression of lck
enhances the Fc
R-induced tyrosine phosphorylation of both ZAP-70
and syk is consistent with the notion that activation of syk family
PTKs is dependent on src family PTKs. These results support and extend
the model proposed by Weiss (52) in which the sequential
activation of TCR-associated PTKs (i.e. src family PTK
proximally, syk family PTK distally) initiates T cell activation. These
studies indicated that a src family PTK is required for the association
of either ZAP-70 or syk with a CD8/
chimera(53, 54) . Similarly, a study conducted with
avian B cells also demonstrates that the presence of a src family PTK
is required for the proper activation of syk after BCR
stimulation(55) . Furthermore, a study with chimeric receptors
of Fc
R (CD16) linked to src and syk family PTKs shows that
clustering of a combination of fyn or lck with ZAP-70 is sufficient to
activate the cytolytic machinery in a cytotoxic T cell
line(56) . Taken together, these studies reflect the tight
linkage that is required between the src and syk families for proper
PTK signaling after receptor activation in lymphoid cells.
R-induced tyrosine
phosphorylation of ZAP-70 than that of syk implies that the regulation
of these two related PTKs may be different. Nonetheless, the presence
of lck significantly enhances both phosphorylation events, suggesting
that the optimal activation of both ZAP-70 and syk is dependent on src
family PTK. This difference between ZAP-70 and syk which we observed in
our experiments is consistent with that of recent reports. Indeed, it
is becoming increasingly evident that antigen receptor-induced syk
activation, but not ZAP-70 activation, can occur in the absence of a
src family PTK. For instance, TCR stimulation of Jurkat T cells lacking
lck can lead to the activation of syk, but not ZAP-70(57) ,
whereas BCR stimulation of B cells lacking detectable src family PTKs
also results in syk activation(55) . In either case, although a
src family PTK is not requisite for syk activation, the presence of a
src family PTK greatly enhances receptor-induced syk
activation(55, 57) . These results correlate with the
earlier study showing that clustering of CD16/syk chimeras by itself is
sufficient to trigger cytolysis, whereas CD16/ZAP-70 chimeras require
coclustering with CD16/fyn chimeras to achieve the same
effect(56) . Regardless of the difference between the two syk
family members, it is clear from our data and those studies by Couture et al.(57) and Kurosaki et al.(55) that optimal activation of syk family PTKs after
receptor stimulation is dependent on the presence of src family PTKs.
R cross-linking in NK cells leads to the activation of
phosphoinositide-specific PLC and the subsequent generation of critical
second messengers(26) . We and others have reported that this is
due to the tyrosine phosphorylation of PLC-
isoforms mediated by
Fc
R-associated PTK(5, 6, 7) . However, the
identity of the PTK responsible for directly phosphorylating PLC-
has remained elusive. The use of the vaccinia virus expression system
allowed us to test whether lck may be involved in coupling the Fc
R
to PLC-
phosphorylation. Our analysis clearly demonstrates that
lck participates in the Fc
R-induced tyrosine phosphorylation of
PLC-
2. This demonstration leaves open the question as to how lck
may be causing the tyrosine phosphorylation of PLC-
2. Increased
PLC-
2 phosphorylation could be due to its direct phosphorylation
by the elevated levels of lck, and this view is supported by the
observation that PLC-
1 can associate with lck in activated T
cells(58) . Alternatively, if lck serves to activate ZAP-70 and
syk, then the phosphorylation of PLC-
2 may be mediated by these
downstream PTKs. In line with this latter hypothesis, clustering of
CD16/syk chimeras alone, or a combination of CD16/ZAP-70 and CD16/fyn
chimeras, can induce the tyrosine phosphorylation of
PLC-
1(56) . Likewise, the targeted disruption of the syk locus in avian B cells, but not the src-related lyn, inhibits the BCR-induced tyrosine phosphorylation of
PLC-
2(51) . Regardless of the precise mechanism utilized,
our results strongly suggest that the activation of lck can couple
Fc
R ligation to the subsequent tyrosine phosphorylation and
regulation of PLC-
2.
R-initiated activation of both
ZAP-70 and syk in NK cells. This interaction between src and syk family
PTKs can have a potent influence on downstream signaling molecules as
evidenced by the effect on PLC-
2. The data presented here provide
the foundation for examining the function of syk family PTKs in
Fc
R signaling in NK cells. In addition, we can also begin to
identify other critical signaling molecules that may be regulated by
lck during NK cell activation. More broadly, these results provide
further insights into the coordinated interaction between multichain
immune recognition receptors and cytoplasmic PTKs.
R, low affinity IgG
Fc receptors (Fc
R type III); PLC, phospholipase C; PTK, protein-
tyrosine kinase; TCR, T cell antigen receptor; BCR, B cell antigen
receptor; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; Tween 20, polyoxyethylene sorbitan monolaurate.
antiserum, Lawrence E. Samelson for providing the
cfb3 peptide, and to Theresa Lee for assistance with the preparation of
this manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.