(Received for publication, October 24, 1994)
From the
To further understand the interactions between Zap-70, Src
family kinases, and other T-cell proteins, we have examined the
regulation of Zap-70 in the antigen-specific T-cell line BI-141. By
analyzing derivatives containing an activated version of either
p56 or p59
, it was
observed that the two Src-related enzymes augmented T-cell receptor
(TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its
association with components of the antigen receptor complex.
Importantly, the accumulation of TCR
Zap-70 complexes
quantitatively and temporally correlated with the induction of tyrosine
phosphorylation of the CD3 and
chains of TCR. Using a
CD4-positive variant of BI-141, we also found that the ability of
Zap-70 to undergo tyrosine phosphorylation and associate with TCR was
enhanced by aggregation of TCR with the CD4 co-receptor. Further
studies allowed the identification of two distinct pools of
tyrosine-phosphorylated Zap-70 in activated T-cells. While one
population was associated with TCR, the other was co-immunoprecipitated
with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In
addition to supporting the notion that Src-related enzymes regulate the
recruitment of Zap-70 in TCR signaling, these data added further
complexity to previous models of regulation of Zap-70. Furthermore,
they suggested that p120 may be an effector and/or a regulator of
Zap-70 in activated T-lymphocytes.
Stimulation of T-lymphocytes by antigen or anti-T-cell receptor
(TCR) ()antibodies results in a rapid rise in intracellular
tyrosine protein phosphorylation (reviewed in Samelson and Klausner,
1992; Veillette and Davidson, 1992; Perlmutter et al., 1993;
Weiss and Littman, 1994). This early biochemical signal leads to a
series of changes that includes elevation of intracellular calcium,
activation of the serine/threonine-specific protein kinase C, and
production of lymphokines such as interleukin (IL)-2 and IL-4.
Ultimately, these modifications allow T-cell mitogenesis and clonal
expansion. The antigen receptor, as well as the associated CD3 and
chains do not possess intrinsic catalytic properties capable of
inducing tyrosine protein phosphorylation. However, the CD3 and
subunits have the ability to promote interactions between cytoplasmic
tyrosine protein kinases and tyrosine phosphorylation substrates,
thereby inducing the TCR signaling cascade.
Accumulating data
indicate that antigen receptor-triggered tyrosine protein
phosphorylation is initiated by p56 and
p59
, two Src-related tyrosine protein kinases
abundantly expressed in T-cells (reviewed in Samelson and Klausner,
1992; Veillette and Davidson, 1992; Perlmutter et al., 1993;
Weiss and Littman, 1994). Upon TCR stimulation, Lck and FynT are
postulated to cause phosphorylation of critical tyrosine residues
located within the tyrosine-based activation motifs of CD3 and
.
This phosphorylation presumably creates docking sites for the
recruitment of molecules involved in signal amplification.
Recently,
significant attention has been directed toward a 70-kDa
tyrosine-phosphorylated protein undergoing association with TCR in
activated T-cells (Chan et al., 1991; Wange et al.,
1992). Through protein purification and cDNA cloning, this polypeptide
(termed Zap-70) was shown to be a novel member of the Syk family of
tyrosine protein kinases, exclusively expressed in T-cells and natural
killer cells (Chan et al., 1992). Like Syk, Zap-70 possesses
two amino-terminal Src homology 2 (SH2) domains, as well as a
carboxyl-terminal catalytic region. Reconstitution experiments in Cos-7
cells have indicated that the association of Zap-70 with TCR requires
signals provided by Lck or FynT (Chan et al., 1992). This idea
is also supported by the finding that TCR stimulation on Jurkat T-cells
failed to provoke association of Zap-70 with TCR in the absence of
p56 expression (Iwashima et al., 1994).
Seemingly, the Src-related enzymes have the ability to trigger binding
of the SH2 domains of Zap-70 to TCR by phosphorylating CD3 and
(Wange et al., 1993; Iwashima et al., 1994;
Timson-Gauen et al., 1994). Finally, as kinase-defective
variants of Zap-70 are still efficiently tyrosine phosphorylated in the
Cos-7 system, the tyrosine phosphorylation of Zap-70 is thought not to
be the result of autophosphorylation. Instead, it is presumed to be
mediated by Src family kinases (Iwashima et al., 1994).
The importance of Zap-70 in normal T-cell physiology has been demonstrated by two lines of experimental evidence. First, Kolanus et al.(1993) reported that antibody-mediated aggregation of chimeric molecules bearing the sequence of Zap-70 linked to the transmembrane and extracellular domains of unrelated molecules is sufficient to cause an elevation of cytoplasmic calcium in T-cells. Hence, Zap-70-mediated signals are likely involved in the regulation of cytosolic calcium during T-cell activation. Second, several groups have demonstrated that the zap-70 gene is mutated in humans with selective T-cell immunodeficiency (Arpaia et al., 1994; Elder et al., 1994; Chan et al., 1994). As Zap-70-deficient individuals exhibit marked alterations in both T-cell differentiation and mature T-cell functions (Arpaia et al., 1994), this observation provides the clearest indication of the central role played by Zap-70 in TCR signaling.
In this report, we have examined the regulation of
Zap-70 in an antigen-specific T-cell line. By studying various BI-141
derivatives, we found that expression of activated forms of
p56 or p59
or
co-aggregation of TCR with CD4 caused an increase in the rapidity and
extent of Zap-70 tyrosine phosphorylation during T-cell activation. The
activated Src-related enzymes also facilitated the association of
Zap-70 with the TCR complex, a likely consequence of their ability to
promote tyrosine phosphorylation of CD3 and
. Evidence was also
obtained that two distinct pools of tyrosine-phosphorylated Zap-70
molecules existed in activated T-cells. While one pool was stably
associated with TCR, the other was complexed with a 120-kDa
tyrosine-phosphorylated protein (p120) of yet unknown identity. In
addition to supporting models of Zap-70 regulation based on studies in
heterologous systems, these data indicated that the regulation of
Zap-70 is more complex than initially suspected. Furthermore, they
raised the possibility that p120 may be an effector and/or a regulator
of Zap-70 during T-lymphocyte activation.
Immune complexes were collected with Staphylococcus aureus protein A (Pansorbin, Calbiochem) coupled, if indicated, to the appropriate second step antibody (either rabbit anti-mouse (RAM) IgG or rabbit anti-hamster IgG). After being recovered by centrifugation, immunoprecipitates were washed several times in lysis buffer, eluted in sample buffer, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE).
To immunoprecipitate the TCR complex, cells were stimulated with MAb F23.1 and RAM IgG. After lysis, immune complexes were collected by the addition of S. aureus protein A (Pansorbin, Calbiochem). Control cells were treated in a similar manner, except that lysis was performed prior to the addition of RAM IgG. For these samples, TCR immunoprecipitation was achieved after supplementing the cell lysates with RAM IgG. In some cases, the TCR-depleted lysates were also subjected to a second round of immunoprecipitation, using standard protocols.
When specified, cells were lysed in boiling 2 TNE
buffer supplemented with 1% SDS to disrupt non-covalent complexes.
After shearing the DNA and removing particulate matters by
centrifugation, lysates were adjusted to a final concentration of 0.1%
SDS, using 1
TNE buffer supplemented with the protease and
phosphatase inhibitors outlined above. Subsequent immunoprecipitations
were conducted as usual.
Figure 1:
Effects of activated p56 and p59
on Zap-70 tyrosine
phosphorylation. Cells were stimulated for the indicated periods of
time with anti-TCR MAb F23.1 and SAM IgG and were then lysed in Nonidet
P-40-containing buffer. A, anti-Zap-70 immunoprecipitations. Toppanel, anti-phosphotyrosine immunoblot; bottompanel, anti-Zap-70 immunoblot. Exposures: toppanel, 16 h; bottompanel, 6 h. B, total cell lysates. Anti-phosphotyrosine immunoblot is
shown. Exposure, 20 h. The positions of prestained molecular mass
markers are shown on the right, while those of p120, Zap-70,
and heavy chain of IgG (Ig) are indicated on the left. Neo, neomycin-resistant
cells.
We
first tested the impact of F505 Lck and F528 FynT on the state of
tyrosine phosphorylation of Zap-70. After incubation with anti-TCR MAb
F23.1, BI-141 cells were activated for variable lengths of time by the
addition of SAM IgG. After lysis in non-ionic detergent-containing
buffer, Zap-70 was immunoprecipitated using a rabbit serum against its
linker region. Zap-70 tyrosine phosphorylation was then ascertained by
anti-phosphotyrosine immunoblotting (Fig. 1A, toppanel). This experiment showed that cells bearing
activated p56 (lanes7-12)
exhibited a marked increase in TCR-induced tyrosine phosphorylation of
Zap-70 when compared with cells expressing the neomycin
phosphotransferase alone (Neo, lanes1-6). Cells containing activated p59
(lanes 13-18) also demonstrated an enhanced Zap-70
tyrosine phosphorylation response, albeit to a lesser extent than cells
expressing F505 Lck. In all cell lines tested, no significant amount of
phosphotyrosine was noted in Zap-70 in the absence of TCR stimulation (lanes1, 7, and 13). Furthermore,
the induction of Zap-70 tyrosine phosphorylation was proportional to
the increase in overall tyrosine protein phosphorylation observed in
the various cell types (Fig. 1B). Importantly, an
anti-Zap-70 immunoblot of Zap-70 immunoprecipitates verified that the
augmented tyrosine phosphorylation of Zap-70 was not due to a change in
its cellular abundance (Fig. 1A, bottompanel).
It should be pointed out that Zap-70
immunoprecipitates from TCR-stimulated cells also contained a
tyrosine-phosphorylated polypeptide of approximately 120 kDa (p120) (Fig. 1A). The accumulation of p120 was clearly greater
in cells containing F505 Lck (lanes 8-12) when compared
with neomycin-resistant cells (lanes 1-6). Moreover, it
tended to be more rapid and more sustained in F528 FynT-expressing
cells (lanes 14-18). The nature of p120 will be further
discussed below. In addition, immunoprecipitates of Zap-70 possessed
tyrosine-phosphorylated products of 85, 78, and 60 kDa. These
polypeptides represent post-translationally modified versions of
Zap-70.
The impact of the activated Src-like enzymes on
the ability of Zap-70 to associate with TCR was also determined. Cells
were stimulated with anti-TCR MAb F23.1 and RAM IgG, as described under
``Materials and Methods.'' Following cell lysis, TCR
complexes were collected by the addition of S. aureus protein
A. After several washes, associated Zap-70 molecules were detected by
immunoblotting with anti-Zap-70 antibodies. Untreated control cells
were lysed immediately after incubation with MAb F23.1 prior to the
addition of the second step antibody. In this case, immunoprecipitation
of TCR was achieved by adding RAM IgG and S. aureus protein A
to the cell lysate. In the absence of TCR stimulation, no or little
Zap-70 was associated with TCR (Fig. 2A, lanes1, 3, 5, 7, 9, and 11). However, upon engagement of TCR, there was easily
appreciable binding of Zap-70 to TCR (lanes2, 4, 6, 8, 10, and 12). In
comparison with expression of the neomycin phosphotransferase alone (lanes 1-4), introduction of activated p56 (lanes 5-8) or activated p59
(lanes 9-12) caused a 10-fold enhancement of the
extent of Zap-70 association with TCR.
Figure 2:
Effects of activated p56 and p59
on Zap-70 association with
TCR. A, effects of activated Lck and FynT on Zap-70
association with TCR. Cells (5
10
) were activated
for 2 min with anti-TCR MAb F23.1 and RAM IgG. After lysis, immune
complexes were collected with S. aureus protein A and were
probed by anti-Zap-70 immunoblotting. Untreated controls were processed
as described under ``Materials and Methods.'' Two different
clones of each type were analyzed in these assays. Lanes1-12, anti-TCR immunoprecipitations; lanes13 and 14, anti-Zap-70 immunoprecipitations
(obtained from 2
10
cells stimulated with MAb F23.1
and SAM IgG). Exposure, 18 h. B, titration. Various numbers of
F505 Lck-expressing cells were activated for 2 min with MAb F23.1 and
SAM IgG (lanes 2-6) or MAb F23.1 and RAM IgG (lanes
8-12). Untreated controls are in lanes1 and 7. Lysates were subjected to either anti-Zap-70 (lanes1-6) or anti-TCR (lanes
7-12) immunoprecipitations and probed by anti-Zap-70
immunoblotting. Exposure, 20 h. The migrations of prestained molecular
size markers are shown on the right, and that of Zap-70 is
indicated on the left. Neo, neomycin-resistant
cells.
To determine the
stoichiometry of these interactions, Zap-70 (Fig. 2B, lanes1-6) and TCR (lanes 7-12)
were individually immunoprecipitated from increasing numbers of MAb
F23.1-stimulated F505 p56-expressing cells. The abundance
of Zap-70 in each of these immunoprecipitates was measured by
anti-Zap-70 immunoblotting and was quantitated using a PhosphorImager.
These data demonstrated that approximately 10% of Zap-70 was complexed
to TCR in activated F505 p56
-bearing cells. This
quantitation is clearly exemplified by comparing the intensity of the
signal in lane12 (which corresponds to 2
10
cells) with those in lanes3 and 4 (which correspond to 0.25
10
and 0.5
10
cells, respectively). A similar proportion of Zap-70
bound the TCR in F528 FynT-bearing cells (Fig. 2A, lanes10 and 12). In contrast, however, only
1% of Zap-70 molecules became associated with TCR in neomycin-resistant
cells (Neo, lanes2 and 4).
Figure 3:
Impact of activated p56 and p59
on tyrosine phosphorylation
of CD3 and
. A, anti-phosphotyrosine immunoblot. Cells
were stimulated for 2 min with anti-TCR MAb F23.1 and RAM IgG, as
described in the legend of Fig. 2A. After
immunoprecipitation, TCR complexes were separated in 12% SDS-PAGE gels
and probed by anti-phosphotyrosine immunoblotting. Lanes1, 3, and 5, untreated controls; lanes2, 4, and 6,
anti-TCR-stimulated cells. Exposure, 6 days. B, time-course
experiment. Cells were activated for the indicated periods of time with
anti-TCR MAb F23.1 and RAM IgG. After lysis, TCR immunoprecipitates
were resolved by 12% SDS-PAGE and probed by immunoblotting with
anti-Zap-70 (toppanel) or anti-phosphotyrosine (bottompanel) antibodies. These two immunoblots were
derived from the same gel. Exposure: toppanel, 22 h; bottompanel, 5 days. The positions of prestained
molecular mass markers are shown on the right, while those of
Zap-70, heavy chain of IgG (Ig), CD3, and
are indicated
on the left. Neo, neomycin-resistant
cells.
These findings suggested that the
ability of activated Src-related enzymes to facilitate the association
of Zap-70 with TCR was indeed related to their efficiency at
phosphorylating CD3/. To obtain further support for this
conclusion, the kinetics and extent of CD3/
tyrosine
phosphorylation were compared with those of Zap-70 binding to TCR.
Thus, TCR immunoprecipitates from resting or activated cells were
immunoblotted with either anti-Zap-70 (Fig. 3B, toppanel) or anti-phosphotyrosine (bottompanel) antibodies. These assays showed that, both in
neomycin-resistant cells (lanes1-6) and in
cells containing F505 p56
(lanes7-12), the onset and extent of Zap-70 association
with TCR closely followed those of tyrosine phosphorylation of
CD3/
. Moreover, the disappearance of TCR-bound Zap-70 (toppanel) clearly coincided with the loss of
tyrosine-phosphorylated CD3/
(bottompanel).
Figure 4:
Regulation of Zap-70 by engagement of the
CD4 co-receptor. A, Zap-70 tyrosine phosphorylation. Cells
were stimulated for 2 min by incubation with the indicated antibodies,
followed by SAM IgG (except for lanes5 and 13, where rabbit anti-rat IgG was used). After lysis, Zap-70
was immunoprecipitated and probed by immunoblotting with either
anti-phosphotyrosine (toppanel) or anti-Zap-70 (bottompanel) antibodies. Lanes9-16 represent total cell lysates. Exposure: toppanel, 20 h; bottompanel, 15
h. B, Zap-70 association with TCR. Cells (5
10
) were stimulated as described for A, except
that RAM IgG was used as a second-step antibody. TCR complexes were
collected with S. aureus protein A and were probed by
anti-Zap-70 immunoblotting. Lanes9 and 10 were obtained from 2
10
cells
immunoprecipitated with anti-Zap-70 antibodies. Exposure, 14 h. The
migrations of prestained molecular size markers are shown on the right, and those of p120, Zap-70, and heavy chain of IgG (Ig) are indicated on the left. Neo,
neomycin-resistant cells.
Changes in Zap-70 tyrosine phosphorylation were monitored by
anti-phosphotyrosine immunoblotting of Zap-70 immunoprecipitates (Fig. 4A, toppanel). Whereas
ligation with anti-TCR MAb F23.1 alone (lane4) or
anti-CD4 MAb GK1.5 alone (lane5) did not cause
tyrosine phosphorylation of Zap-70, co-aggregation of TCR and CD4 (lane6) promptly increased the phosphotyrosine
content of Zap-70 in a manner analogous to expression of F505
p56 (lane8). Quantitative analyses of
these data indicated that Zap-70 was seven times more tyrosine
phosphorylated after co-aggregation of TCR and CD4 (lane6), when compared with stimulation of TCR alone (lane4). Co-ligation of TCR with CD4 also increased the
recovery of tyrosine-phosphorylated p120 by approximately 5-fold (lane6).
The extent of association of Zap-70 with TCR was also determined (Fig. 4B). Antibody-mediated co-aggregation of TCR and CD4 significantly enhanced the binding of Zap-70 to TCR (lane6). Indeed, while essentially no detectable Zap-70 was bound to TCR in MAb F23.1-stimulated cells (lane4), approximately 5% of these molecules became associated with TCR in cells treated with MAb F23.1-MAb GK1.5 heteroconjugates (lane6).
Figure 5: Identification of two populations of tyrosine-phosphorylated Zap-70 in activated BI-141 T-cells. Cells were stimulated with anti-TCR MAb F23.1 and RAM IgG and lysed, and TCR complexes were immunoprecipitated by the addition of S. aureus protein A. TCR-depleted lysates were subjected to re-immunoprecipitation with anti-Zap-70 antibodies. Immunoprecipitates were blotted with either anti-phosphotyrosine (toppanels) or anti-Zap-70 (bottompanels) antibodies. A, characterization of cells expressing F505 Lck and F528 FynT. All cells were stimulated for 2 min with anti-TCR MAb F23.1 and RAM IgG. Exposures: toppanel, 16 h; bottompanel, 16 h. B, time course of TCR stimulation on F505 Lck-expressing cells. Exposures: toppanel, 16 h; bottompanel, 14 h. The positions of prestained molecular mass markers are shown on the right, and those of p120, Zap-70, and heavy chain of IgG (Ig) are indicated on the left. Neo, neomycin-resistant cells.
Because the association
of CD3/ with TCR can be dissociated by non-ionic detergents such
as Nonidet P-40, we wished to verify that the detection of a
non-TCR-bound pool of tyrosine-phosphorylated Zap-70 was not consequent
to artificial dissociation of CD3/
from TCR. To this end,
TCR-depleted lysates were re-immunoprecipitated with antibodies
directed against Zap-70 (Fig. 6, lane3),
(lanes4 and 5), or CD3-
(lane6), and recovered polypeptides were probed by
anti-phosphotyrosine immunoblotting. While abundant quantities of
phosphotyrosine-containing Zap-70 were present in TCR-depleted lysates (lane3, toppanel), these lysates
contained little or no tyrosine-phosphorylated CD3 or
(lanes4, 5, and 6, bottompanel). Since the association of Zap-70 with CD3/
is
known to be resistant to the presence of Nonidet P-40 (Chan et
al., 1991), this observation implied that the accumulation of
tyrosine-phosphorylated Zap-70 in TCR-depleted lysates was not due to
post-lysis dissociation of CD3/
from TCR.
Figure 6:
Characterization of TCR-depleted lysates.
TCR-depleted lysates from F505 p56-containing
cells were re-immunoprecipitated with the indicated antibodies. After
separation in 12% SDS-PAGE gels, polypeptides were detected by
immunoblotting with anti-phosphotyrosine antibodies. The top and bottompanels correspond to different
exposures of the same immunoblot. The absence of a detectable heavy
chain of IgG in lane5 is due to the fact that MAb
H146 does not require a second-step antibody for binding S. aureus protein A. The 28-kDa immunoreactive product present in
anti-Zap-70 immunoprecipitates (lane3) is seemingly
a degradation product of Zap-70.
Exposures: toppanel, 16 h; bottompanel, 6 days. The
positions of prestained molecular mass markers are shown on the right, and those of p120, Zap-70, heavy chain of IgG (Ig), CD3, and
are indicated on the left.
Figure 7:
Characterization of p120. F505
p56-bearing cells were stimulated for 2 min with
anti-TCR MAb F23.1 and SAM IgG. Immunoprecipitates were then analyzed
by anti-phosphotyrosine immunoblotting. A, specificity of
co-immunoprecipitation of Zap-70 and p120. Exposure, 16 h. B,
p120 is co-immunoprecipitated by multiple anti-Zap-70 sera. Exposure, 4
days. C, effects of denaturation on the Zap-70-p120
interaction. Cells were stimulated for the indicated periods of time
with anti-TCR MAb F23.1 and SAM IgG. They were then lysed in either
standard Nonidet P-40-containing lysis buffer (lanes1-6) or in boiling SDS-containing buffer (lanes
7-12). Exposure, 2 days. D, effects of
phenylphosphate on the Zap-70-p120 interaction. After lysis in Nonidet
P-40-containing buffer, Zap-70 immunoprecipitations were conducted in
the presence of progressively higher concentrations of phenylphosphate (lanes1-6) or phosphoserine (lanes7-12). Exposure, 2 days. The positions of
prestained molecular mass markers are shown on the right,
while those of p120, Zap-70, and heavy chain of IgG (Ig) are
indicated on the left.
Two independent rabbit antisera generated against the linker region of Zap-70, as well as a polyclonal rabbit serum recognizing the SH2 domains of Zap-70, were evaluated in our assays (Fig. 7B). The two anti-linker sera were equally efficient at recovering the tyrosine-phosphorylated p120 (lanes2 and 4). The antiserum reacting with the Zap-70 SH2 domains also precipitated p120 (lane6), albeit at a lower efficiency. This serum was also less adequate at immunoprecipitating Zap-70 (lane6; data not shown). Similar data were obtained using an antiserum against the carboxyl terminus of Zap-70 (data not shown). Perhaps the antibodies directed against the linker region (which lies between the end of the second SH2 domain and the beginning of the catalytic region) stabilized the interaction of Zap-70 with p120, thus facilitating its recovery. Alternatively, the anti-SH2 domains and anti-carboxyl terminus sera may have lowered the immunoprecipitation of p120 by causing its displacement from Zap-70.
These results implied that p120 specifically interacted with Zap-70 in activated BI-141 cells. To better understand the basis for this interaction, the effects of denaturation were examined (Fig. 7C). Cells were lysed either in standard Nonidet P-40-containing buffer (lanes1-6) or in boiling SDS-containing buffer (lanes7-12) prior to immunoprecipitation with anti-Zap-70 antibodies. An anti-phosphotyrosine immunoblot of these immunoprecipitates revealed that the presence of SDS completely abolished the association of p120 with Zap-70 (lanes7-12). Although the ability to immunoprecipitate Zap-70 was also partially reduced in the SDS-containing buffer, this diminution was probably consequent to the added ionic strength provided by the detergent.
The potential involvement of tyrosine-phosphorylated residues in the formation of this complex was also evaluated (Fig. 7D). Zap-70 was immunoprecipitated in the absence or presence of progressively higher concentrations of either phenylphosphate, an analog of phosphotyrosine (lanes2-6), or phosphoserine (lanes 8-12). A subsequent anti-phosphotyrosine immunoblot showed that phenylphosphate caused a dose-dependent dissociation of p120 from Zap-70 (lanes 2-6), which was nearly complete at a concentration of 50 mM (lane5). A similar effect was obtained with phosphotyrosine (data not shown). In contrast, however, equivalent concentrations of phosphoserine failed to interfere with the binding of p120 to Zap-70 (lanes 8-12).
In
an attempt to identify p120, lysates were immunoprecipitated with
antibodies against known tyrosine phosphorylation substrates of
approximately 120 kDa. The phosphotyrosine content of these products in
activated BI-141 cells was then measured by anti-phosphotyrosine
immunoblotting (Fig. 8). No significant tyrosine phosphorylation
was detected in immunoprecipitates of the 110-kDa phosphatidylinositol
3` kinase (precipitated with anti-p85 antibodies) (lane3), the 110-kDa actin-binding protein p110 (lane4), the 120-kDa GTPase-activating protein of p21 (lane5), and the integrin-regulated tyrosine
protein kinase p125
(lanes6 and 7). Recent data also demonstrated that p120
,
the cellular homolog of the Cbl oncoprotein, undergoes prominent
tyrosine phosphorylation in activated Jurkat T-cells (Donovan et
al., 1994). While anti-Cbl antibodies also identified a prominent
120-kDa tyrosine-phosphorylated protein in activated BI-141 cells (Fig. 8B, lane3), this polypeptide
was not detectably associated with Zap-70. Based on this result, it
appeared unlikely that the 120-kDa Zap-associated protein was Cbl.
Figure 8:
The Zap-70-associated p120 is distinct
from known tyrosine phosphorylation substrates. F505
p56-bearing cells were stimulated for 2 min with
anti-TCR MAb F23.1 and SAM IgG. Various immunoprecipitates were then
analyzed by anti-phosphotyrosine immunoblotting. Exposures, 17 h. NRS,
normal rabbit serum.
In this paper, we have studied the regulation of Zap-70 in
the antigen-specific T-cell line BI-141. We found that expression of
activated versions of p56 or p59
caused an
appreciable increase in the rapidity, intensity, and duration of Zap-70
tyrosine phosphorylation during T-cell activation (Fig. 1A). Furthermore, using CD4-positive BI-141
derivatives, it was observed that antibody-mediated aggregation of TCR
with the CD4 co-receptor allowed a greater accumulation of
tyrosine-phosphorylated Zap-70 (Fig. 4A). As ligation
of CD4 is known to activate p56
(Veillette et
al., 1989; Luo and Sefton, 1990), this observation further
supported the notion that the recruitment of Zap-70 in TCR signaling is
regulated by Src-like enzymes.
We also demonstrated that activated
Lck and FynT, as well as co-ligation of TCR with CD4, increased the
extent of Zap-70 association with TCR by 5-10-fold ( Fig. 2and Fig. 4B). Importantly, this
enhancement paralleled the ability of Lck and FynT to promote CD3 and
tyrosine phosphorylation (Fig. 3). Indeed, in careful
time-course analyses, it was shown that the accumulation of
TCR
Zap-70 complexes in activated T-cells coincided with the
induction of CD3/
tyrosine phosphorylation.
Given the
sensitivity of the anti-Zap-70 antibodies used in our studies, we were
able to obtain an assessment of the stoichiometry of association of
Zap-70 with TCR. Under linear assay conditions, it was estimated that
roughly 1% of Zap-70 molecules became bound to TCR in activated
neomycin-resistant BI-141 T-cells. This proportion rose to 10% in
cells bearing activated versions of p56
or
p59
. Furthermore, in CD4-positive BI-141 derivatives,
approximately 5% of Zap-70 polypeptides became associated with TCR upon
co-aggregation of TCR with CD4.
Together, these observations
supported the model proposed by others (Chan et al., 1992;
Iwashima et al., 1994; Timson-Gauen et al., 1994),
which was primarily based on reconstitution experiments in non-lymphoid
cells. Seemingly, engagement of TCR triggers the T-cell activation
cascade by allowing Lck and/or FynT to phosphorylate CD3/. This
phosphorylation allows the recruitment of Zap-70 at the membrane, where
it is susceptible to phosphorylation by Src-related enzymes.
Membrane-associated and tyrosine-phosphorylated Zap-70 is presumably
able to phosphorylate its putative targets and/or to behave as a
docking molecule, capable of recruiting additional signal transducers
at the plasma membrane.
These concepts are somewhat complicated by
the finding that two distinct pools of tyrosine-phosphorylated Zap-70
molecules were present in activated BI-141 cells (Fig. 5).
Whereas one pool was stably associated with TCR, thus fulfilling the
scheme described above, a second more prominent population was not
detectably associated with TCR. In light of this observation, special
efforts were made to rule out the possibility that the non-TCR-bound
Zap-70 was produced as a result of artificial dissociation of CD3/
from TCR (Fig. 6). However, we felt confident that our TCR
immunoprecipitates contained essentially all the
tyrosine-phosphorylated CD3/
molecules from activated BI-141
cells. Coupled with the fact that the interaction between CD3/
and
Zap-70 is not affected by lysis conditions similar to those used in our
studies (Chan et al., 1991), it thus appeared unlikely that
the existence of non-TCR-associated tyrosine-phosphorylated Zap-70 was
consequent to in vitro disruption of the antigen receptor
complex.
The mechanisms of production, as well as the functions of
these two groups of tyrosine-phosphorylated Zap-70 remain to be
clarified. Our kinetics analyses indicated that the presence of
TCR-associated tyrosine-phosphorylated Zap-70 was transient in
activated T-cells (Fig. 5B). In contrast, the
accumulation of non-TCR-associated polypeptides tended to be more
sustained. Because tyrosine phosphorylation of both pools of Zap-70 was
increased by activated Src-related enzymes (Fig. 5A),
it is conceivable that they had a common origin. Possibly, the
non-TCR-associated population was generated following in vivo dissociation of the TCR-bound pool, perhaps as a consequence of
CD3/ dephosphorylation. Although it seems somewhat less likely, it
remains also possible that the two populations of Zap-70 were produced
through distinct processes. Future studies will be necessary to test
these possibilities.
Interestingly, we noted that non-TCR-associated
Zap-70 molecules from activated BI-141 cells specifically
co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated
polypeptide. In addition to lending credence to the notion that the two
pools of Zap-70 may serve distinct functions during TCR signaling, this
finding intimated that p120 may be either a substrate or a regulator of
Zap-70. Unfortunately, however, we have not yet been able to define the
identity of p120. Our results indicated that the Zap-70-associated p120
was not the catalytic subunit of phosphatidylinositol 3` kinase,
GTPase-activating protein, p125, or the 110-kDa
actin-binding protein (p110) (Fig. 8A). Furthermore, it
did not appear to be Cbl, a 120-kDa polypeptide recently shown to
undergo prominent tyrosine phosphorylation in activated T-lymphocytes (Fig. 8B) (Donovan et al., 1994). While not
formally excluded, it is also improbable that p120 was a protein
kinase, as it failed to undergo phosphorylation during immune complex
kinase reactions.
Additional biochemical
characterizations showed that the Zap-70p120 complex was
dissociated by boiling in the presence of SDS, implying that it was
non-covalent in nature (Fig. 7C). Moreover, it could be
disrupted by either phenylphosphate (Fig. 7D) or
phosphotyrosine (data not shown), suggesting that it was dependent on
the presence of phosphorylated tyrosine residues. However, the complex
did not apparently involve the SH2 domains of Zap-70, as recombinant
fusion proteins bearing this portion of Zap-70 could not recover
tyrosine-phosphorylated p120 from activated T-cell lysates.
Therefore, the putative critical sites of tyrosine
phosphorylation may be situated on Zap-70 itself. Obviously, further
characterization of p120 is likely to provide useful insights into the
regulation and function(s) of Zap-70 in T-lymphocytes.
A recent
report suggested that Syk, a Zap-70-related tyrosine protein kinase
expressed in at least some T-cells, also plays a prominent role in
T-cell receptor signaling (Couture et al., 1994). It was
observed that, unlike Zap-70, Syk is constitutively associated with
TCR. Furthermore, the authors reported that TCR stimulation caused an
activation of the catalytic function of Syk. They proposed that
Syk-mediated signals during T-cell activation may be more proximal than
those provided by Src-related enzymes or Zap-70. While it is possible
that this scheme applies to a subset of T-cells, it should be pointed
out that we have been unable to detect expression of Syk in BI-141
T-cells, using at least two different anti-Syk sera. Hence,
based on our data, Syk is clearly not an essential component of the TCR
signaling pathway.
In summary, we have shown that the ability of
Zap-70 to undergo tyrosine phosphorylation and associate with TCR is
markedly enhanced by expression of activated forms of p56 and p59
or by ligation of TCR with the CD4
co-receptor. The extent and duration of Zap-70 association with TCR
correlated with those of tyrosine phosphorylation of CD3 and
,
thus supporting the notion that Src-related enzymes initiate
TCR-mediated signals by catalyzing the tyrosine phosphorylation of
these TCR components. Our studies also allowed the identification of
two distinct pools of tyrosine-phosphorylated Zap-70 in activated
T-cells. Whereas one pool was associated with TCR, the other was devoid
of demonstrable interactions with this structure. Even though the
nature and function of these two populations were not determined, it
was observed that the non-TCR-associated fraction specifically
co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated
polypeptide of yet undefined identity. These data definitely add
further complexity to previous models of regulation of Zap-70.
Furthermore, they suggest that p120 may be a target and/or a regulator
of Zap-70 in activated T-lymphocytes.