(Received for publication, June 24, 1994; and in revised form, September 9, 1994)
From the
Signaling by the T cell antigen receptor (TCR) is mediated by
17-residue tyrosine-based activation motifs (TAM) present in the
cytoplasmic tails of the TCR and CD3 chains. TAMs become
tyrosine-phosphorylated upon TCR stimulation, creating a high affinity
binding site for the tandem SH2 domains of ZAP-70. In permeabilized T
cells, the association of TCR and ZAP-70 was inhibited by a protein
tyrosine phosphatase (PTPase)-resistant TAM peptide analog, in which
difluorophosphonomethyl phenylalanyl (F
Pmp) residues
replaced phosphotyrosine. Inhibition of this association prevented
TCR-stimulated tyrosine phosphorylation of ZAP-70 and reduced ZAP-70
kinase activity to basal levels. The reduction in ZAP-70 activity
coincided with reduced tyrosine phosphorylation of a number of
substrates. Such PTPase-resistant peptides, capable of disrupting SH2
domain-mediated protein-protein interactions, should prove useful in
further dissection of multiple signaling pathways and may serve as
models for rationally designed chemotherapeutic agents for the
treatment of autoimmune and neoplastic disorders.
The T cell-mediated immune response depends upon the ability of
the T cell antigen receptor (TCR) ()to recognize specific
antigen and respond by initiating an intracellular signaling cascade.
The
and
chains of the multisubunit TCR determine the
antigen specificity of the TCR, while the
,
, and
CD3
chains and the TCR
chain are coupled to intracellular signaling
molecules. A number of studies have demonstrated the importance of a
short amino acid sequence, present as three copies in the cytoplasmic
domain of the TCR
chain and as a single copy in each of the CD3
chains, for initiating T cell
signaling(1, 2, 3) . This sequence has many
descriptive names including the tyrosine-based activation motif (TAM)
and has the general structure
YXX(L/I)X
YXX(L/I) (4) . In chimeric receptor cross-linking studies, TAMs have
been shown to be necessary and sufficient for initiation of T cell
activation (1, 2, 3) .
The earliest
biochemical event detected upon TCR stimulation is increased tyrosine
phosphorylation of a number of cellular substrates (5) ,
including the TCR chain TAMs(6, 7, 8) ,
ZAP-70(9) , Vav(10, 11) , Shc(12) , phospholipase
C1(13, 14) , and VCP (15) . Many other
substrates have yet to be identified. Three protein tyrosine kinases
that have been implicated as being involved in this early phase of
TCR-mediated signaling are the Src-family members Fyn and Lck and the Syk-family member ZAP-70. The
evidence for the involvement of these protein tyrosine kinases in TCR
signaling has been extensively reviewed (16, 17, 18) .
Recently it was shown that Lck and ZAP-70 can act sequentially to tyrosine phosphorylate
various substrates in COS cells transfected with Lck, ZAP-70,
and a CD8 chimeric protein (19) . In this system, Lck is required to tyrosine phosphorylate the TAMs of CD8
which
permits ZAP-70 binding. Upon association with CD8
, ZAP-70 is
tyrosine-phosphorylated, becomes activated, and induces tyrosine
phosphorylation of substrates. One would predict from such a model that
agents that block the association of ZAP-70 with the TCR could prove
useful in identifying the downstream effectors of ZAP-70 and in
blocking T cell activation. Recent insights into the mechanism of the
association of ZAP-70 with the TCR provide the basis for the design of
such inhibitory agents.
Src-homology 2 (SH2) domains
mediate protein-protein interactions by binding to pTyr-containing
sequences(20, 21) . The presence of two SH2 domains in
ZAP-70 and 2 pTyr residues in stimulated TCR TAMs suggested a possible
mechanism of interaction. Experiments using glutathione S-transferase fusion proteins containing the SH2 domains of
ZAP-70 show that ZAP-70 binds to the tyrosine-phosphorylated TCR
and CD3
chains via its SH2 domains; both SH2 domains are required
for high affinity binding(22) . That the TCR TAM motifs mediate
this interaction was demonstrated in studies showing binding of ZAP-70
to transmembrane chimeric receptors with a single
chain TAM as
the cytoplasmic tail(3) . In this study, we report that a novel
phosphatase-resistant synthetic peptide based on the C-terminal
(pTyr)
-TAM of the TCR
chain competitively blocks
ZAP-70 binding to activated TCR. As a consequence, TCR stimulation
fails to induce tyrosine phosphorylation of ZAP-70 or activation of its
kinase activity. Coincident with this loss of ZAP-70 kinase activity is
reduced tyrosine phosphorylation of a number of substrates. This result
suggests that the peptide blocks early TCR-mediated signaling events
through a ZAP-70 dependent mechanism, as would be predicted by the
model of early activation events mentioned above.
Because of the presence of active protein tyrosine
phosphatases (PTPases) in T
cells(31, 32, 33) , the peptides were
synthesized with a phosphotyrosyl analog that is resistant to
hydrolysis. In this analog, difluorophosphonomethyl phenylalanine
(FPmp), a difluoromethylene group replaces the phenolic
oxygen of phosphotyrosine (24, 25) . In previous
studies, F
Pmp- and pTyr-containing hexapeptides have been
shown to bind their complementary SH2 domains with similar
affinities(34) . Four different F
Pmp-containing
peptides were synthesized based on the sequence LYQGLSTATKDTYDALH,
which is the C-terminal (third) TAM of the human TCR
chain. The
peptide in which both Tyr residues are replaced with F
Pmp
groups is referred to as
(F
Pmp)
-TAM
, while peptides in
which either the N- or C-terminal Tyr is replaced with F
Pmp
are (F
Pmp)
-TAM
or
(F
Pmp)
-TAM
, respectively. The
fourth peptide is a control in which the phosphonate oxygens of
(F
Pmp)
-TAM
remain blocked with
ethoxy groups from the synthesis.
The ability of these synthetic
peptides to bind to ZAP-70 was assessed in competition binding studies
with a synthetic P-labeled
(pTyr)
-TAM
peptide that binds a
glutathione S-transferase fusion protein containing both SH2
domains of ZAP-70 (Fig. 1). Only those peptides containing two
pTyr or F
Pmp groups competed for binding. The ether-blocked
control peptide did not compete. No peptide binding was observed to
glutathione S-transferase alone or fusion proteins containing
a single C- or N-terminal ZAP-70 SH2 domain (not shown).
(F
Pmp)
-TAM
bound the tandem
SH2 domains of ZAP-70 with an affinity similar to the
(pTyr)
-TAM
itself. A higher concentration
of cold (pTyr)
-TAM
(1 µM) was
required to give 50% competition of 4 nM
P-labeled (pTyr)
-TAM
.
This disparity reflects the fact that the effective concentration of
the binding site, glutathione/agarose-adsorbed glutathione S-transferase-ZAP-70-(SH2)
, is much higher than
that of the labeled peptide. The excess of binding sites over ligand in
this assay precludes the determination of absolute affinities from the
IC
values, but these values remain useful in deriving
relative affinities(35) . In additional binding studies,
(F
Pmp)
-TAM
cross-competed with
(pTyr)
-TAM
,
(pTyr)
-TAM
, and
(pTyr)
-TAM
indicating that ZAP-70 binding to all
TCR
and CD3
TAMs can be inhibited by
(F
Pmp)
-TAM
.
Figure 1:
Competitive binding of
FPmp-containing peptides to glutathione S-transferase-ZAP-70(SH2)
. Three µg of
glutathione S-transferase-ZAP-70-(SH2)
immobilized
on glutathione agarose were incubated with
-
P-labeled
(pTyr)
-TAM
(4 nM) together with
increasing concentrations of the indicated peptides. After 1 h of
incubation at 4 °C, the beads were washed extensively, and bound
radioactivity was measured by scintillation counting.
,
(F
Pmp)
-TAM
;
,
(F
Pmp)
-TAM
;
,
(F
Pmp)
-TAM
;
, control
peptide;
, (pTyr)
-TAM
. Data are from
one experiment and are representative of three
experiments.
Having found that
(FPmp)
-TAM
could bind ZAP-70 in vitro, the ability of
(F
Pmp)
-TAM
to bind ZAP-70 and
block its association with TCR was tested in Jurkat T cells
permeabilized with the bacterial toxin tetanolysin. The TCR from
peptide-treated permeabilized Jurkat T cells was immunoprecipitated and
examined for associated ZAP-70, as detected by anti-ZAP-70 or anti-pTyr
blotting (Fig. 2A). As demonstrated previously, ZAP-70
bound the TCR only upon TCR stimulation(9, 23) . The
association of ZAP-70 with TCR was markedly inhibited by
(F
Pmp)
-TAM
, but not by the
ether-blocked control peptide or the singly
F
Pmp-substituted peptides. The ZAP-70 co-precipitated with
TCR was tyrosine-phosphorylated (Fig. 2A). In addition,
co-recovery of phospho-
or phospho-
with ZAP-70
immunoprecipitates was also inhibited by
(F
Pmp)
-TAM
(not shown).
(F
Pmp)
-TAM
could be acting to
block co-recovery of ZAP-70 with TCR either by blocking initial
TCR-stimulated binding or by competing after cell lysis. To test the
latter possibility, the peptide was added after termination of TCR
stimulation, such that the peptide could not block initial assembly,
but could compete with the TCR for ZAP-70 during immunoprecipitation.
Only a minimal reduction in ZAP-70 co-recovery with the TCR was
observed, suggesting that the major effect of
(F
Pmp)
-TAM
is to block initial
assembly of ZAP-70 with the TCR (not shown).
Figure 2:
(FPmp)
-TAM
inhibition of TCR-stimulated ZAP-70/TCR association and ZAP-70 tyrosine
phosphorylation. A, TCR was immunoprecipitated from
unstimulated(-) or TCR-stimulated (+)
tetanolysin-permeabilized Jurkat T cells incubated with 4 µM concentrations of the indicated peptides. The immunoprecipitates
were analyzed for co-precipitating ZAP-70 with a mAb to pTyr (4G10) (upper panel) or a polyclonal antiserum to ZAP-70 (lower
panel). B, ZAP-70 was immunoprecipitated from intact
Jurkat T cells and Jurkat T cells treated as in A. The
immunoprecipitates were analyzed for ZAP-70 with 4G10 (upper
panel) or a polyclonal antiserum to ZAP-70 (lower
panel).
Recent studies would
predict that inhibition of ZAP-70 association with the TCR would reduce
TCR-stimulated tyrosine phosphorylation of ZAP-70 (19) . To
test this prediction, anti-ZAP-70 immunoprecipitates from permeabilized
Jurkat T cells, incubated with
(FPmp)
-TAM
, were blotted for
pTyr (Fig. 2B).
(F
Pmp)
-TAM
blocked the
TCR-stimulated tyrosine phosphorylation of total ZAP-70, while the
control peptide and the singly F
Pmp-substituted peptides
did not. The amount of ZAP-70 immunoprecipitated was the same in each
lane as shown in the anti-ZAP-70 blot of a replicate gel (Fig. 2B). The same effect of
(F
Pmp)
-TAM
on ZAP-70 tyrosine
phosphorylation was also seen in anti-pTyr blots of whole cell lysates
from peptide-treated permeable Jurkat cells (see Fig. 4A). The inhibitory effect of
(F
Pmp)
-TAM
on ZAP-70 tyrosine
phosphorylation is not mediated by activation of a PTPase activity,
since this effect is unaffected by orthovanadate (not shown).
Figure 4:
(FPmp)
-TAM
inhibition of tyrosine phosphorylation of Jurkat T cell substrates. A, whole cell lysates were collected from
unstimulated(-) or TCR-stimulated (+)
tetanolysin-permeabilized Jurkat T cells incubated without added
peptide(-), with control peptide (C), or with
(F
Pmp)
-TAM
(T).
Lysates were also collected from unstimulated(-) or
TCR-stimulated (+) intact Jurkat T cells. Lysates were analyzed
for pTyr-containing proteins by immunoblotting with 4G10. The arrowhead indicates the position of ZAP-70. The arrow shows the position of TCR chains. The dots indicate the
position of other proteins with reduced tyrosine phosphorylation upon
(F
Pmp)
-TAM
incubation. B, TCR was immunoprecipitated as in Fig. 2, except that
cells were also incubated with (+) or without(-) 1 mM orthovanadate. TCR subunits were analyzed for pTyr by 4G10
immunoblotting.
The
effect of (FPmp)
-TAM
incubation on ZAP-70 kinase activity was determined in an immune
complex kinase assay. Since no substrates of the ZAP-70 kinase have yet
been identified, we used a cytosolic fragment of the erythrocyte band 3
protein (cfb3) as a substrate in the kinase assay(36) . cfb3 is
known to be a substrate for the related protein tyrosine kinase, Syk(37) . ZAP-70 was immunoprecipitated from lysates
of intact or permeabilized cells incubated with and without
(F
Pmp)
-TAM
. The immune complex
kinase assay was carried out on a portion of the immunoprecipitated
ZAP-70, while the remainder was analyzed for the pTyr content of ZAP-70
by anti-pTyr blotting (Fig. 3, upper and middle
panels, respectively). Stimulation of the TCR by mAb ligation
increased the kinase activity associated with ZAP-70, as reflected in
increased phosphorylation of cfb3 and ZAP-70 itself. The predominant
kinase activity associated with the ZAP-70 immunoprecipitate is ZAP-70
itself, although other kinases that associate with either ZAP-70 or
phospho-
could also contribute activity in this assay. Two kinases
that would be most likely to co-precipitate with ZAP-70 or associated
molecules are Lck and Syk(38, 39, 40) ; however, neither Lck activity nor Syk protein is detected in the ZAP-70
immunoprecipitates
(41) , suggesting that the kinase
activity observed is predominantly due to ZAP-70. Incubation of
permeabilized Jurkat with (F
Pmp)
-TAM
reduced the kinase activity of ZAP-70 to the level observed in
unstimulated intact cells, while control peptide had no effect. The
level of tyrosine phosphorylation of the ZAP-70 used in the kinase
assay shows that the basal kinase activity is associated with
nonphosphorylated ZAP-70, while increased kinase activity is associated
with tyrosine-phosphorylated ZAP-70 (Fig. 3, middle
panel). The amount of ZAP-70 in each lane was the same, as is
shown by anti-ZAP-70 blotting (lower panel). The actual
mechanism of ZAP-70 tyrosine phosphorylation remains to be determined,
and, while ZAP-70 activation coincides with both TCR binding and
tyrosine phosphorylation of ZAP-70, it is still unclear which, if
either, of these events activates the kinase.
Figure 3:
(FPmp)
-TAM
inhibition of TCR-stimulated ZAP-70 kinase activity. ZAP-70 was
immunoprecipitated from TCR-stimulated (+),
tetanolysin-permeabilized Jurkat T cells incubated without peptide, or
with either the (F
Pmp)
-TAM
(T) or the ether-blocked control peptide (C).
For comparison, ZAP-70 was also immunoprecipitated from
unstimulated(-) or TCR-stimulated (+) intact Jurkat T cells.
The immunoprecipitates were analyzed for in vitro kinase
activity (upper panel). A portion of the immunoprecipitates
were analyzed for tyrosine-phosphorylated ZAP-70 by 4G10 immunoblotting (middle panel). The relative amount of ZAP-70 in each
immunoprecipitate was analyzed by immunoblotting the nitrocellulose
membrane from the kinase assay (upper panel) with a polyclonal
antiserum to ZAP-70 (lower panel).
The results show a
direct correlation between
(FPmp)
-TAM
-induced inhibition
of ZAP-70 association with activated TCR, loss of tyrosine
phosphorylation of ZAP-70, and loss of TCR-stimulated ZAP-70 kinase
activity. These findings are consistent with a model whereby
(F
Pmp)
-TAM
binds to ZAP-70, occupies the
tandem SH2 domains of ZAP-70, and thereby prevents ZAP-70 association
with tyrosine-phosphorylated TCR. Inhibition of ZAP-70 binding to the
TCR then prevents tyrosine phosphorylation and activation of ZAP-70. An
alternate model in which (F
Pmp)
-TAM
binds to Lck, prevents Lck from
tyrosine-phosphorylating the TCR, and thus prevents association of
ZAP-70 with the TCR, is unlikely, since Lck does not bind a
synthetic peptide with a similar structure to
(F
Pmp)
-TAM
(19) . Also,
elimination of the binding capacity of the SH2 domain of Lck by mutation does not affect its ability to tyrosine-phosphorylate
or ZAP-70(19) . In addition, the
F
Pmp-containing peptides have no effect on Lck kinase activity toward a synthetic peptide, containing residues
52-164 of the cytoplasmic tail of
in an in vitro kinase assay
(27) .
To determine whether
(FPmp)
-TAM
-mediated inhibition
of ZAP-70 association with the TCR had an effect on TCR-stimulated
tyrosine phosphorylation of Jurkat T cell substrates, lysates from
Jurkat T cells were treated as indicated in Fig. 4and blotted
for pTyr. In intact cells there was a marked increase in tyrosine
phosphorylation of many proteins in response to mAb stimulation of the
TCR. Although tetanolysin permeabilization, itself, caused increased
tyrosine phosphorylation of some proteins, stimulation of the TCR in
the permeabilized cells caused a further increase in tyrosine
phosphorylation. Addition of
(F
Pmp)
-TAM
decreased tyrosine
phosphorylation of a number of proteins, while control peptide had no
effect. As was seen in Fig. 2B, the
(F
Pmp)
-TAM
peptide almost
completely blocked tyrosine phosphorylation of ZAP-70. Several other
unidentified proteins with molecular masses of 120, 80, and 35-40
kDa showed reduced tyrosine phosphorylation after incubation with
(F
Pmp)
-TAM
. The identification
of these phosphoproteins, which might be ZAP-70 substrates, and the
effect of (F
Pmp)
-TAM
on distal
signaling molecules in viable T cells are the subjects of continuing
investigation. Jurkat T cells have recently been shown to contain the
protein tyrosine kinase Syk(40, 41) . In these cells,
Syk is recruited to phospho-
concurrently with
ZAP-70(41) . Thus, it remains formally possible that part of
the effect of (F
Pmp)
-TAM
on
substrate phosphorylation may be due to the blockade of Syk association
with the TCR.
Tyrosine phosphorylation of and CD3
receptor chains was also decreased in
(F
Pmp)
-TAM
-treated Jurkat T
cells (Fig. 4A). If our model of
(F
Pmp)
-TAM
action is correct,
then reduced tyrosine phosphorylation of
and CD3
is not
likely to be due to reduced kinase activity, as ZAP-70 exhibits no
activity for TCR subunits, and
(F
Pmp)
-TAM
fails to block Lck activity
(19) . One possibility to
explain the decreased TCR tyrosine phosphorylation is that
(F
Pmp)
-TAM
could increase the effective
PTPase activity toward the TCR by blocking the ability of ZAP-70 to
bind to and protect the TAM pTyr residues from
dephosphorylation(19) . Such a mechanism has been demonstrated
for phospholipase C
1 binding to the epidermal growth factor
receptor(42) . To test this possibility, the TCR was
immunoprecipitated from peptide-treated Jurkat T cells in the presence
or absence of orthovanadate during TCR stimulation and analyzed by
anti-pTyr blotting (Fig. 4B). Orthovanadate increased
the overall pTyr signal, but also inhibited the ability of the
(F
Pmp)
-TAM
peptide to reduce
tyrosine phosphorylation of
and CD3
. This suggests that, in
the absence of orthovanadate, the TAMs are tyrosine-phosphorylated
normally, but are dephosphorylated rapidly in the absence of ZAP-70
binding. Orthovanadate did not block the
(F
Pmp)
-TAM
-mediated reduction
in tyrosine phosphorylation of other substrates (not shown). Therefore,
we favor the interpretation that the principle action of
(F
Pmp)
-TAM
is to bind to
ZAP-70 and block its association with the TCR. Failure of ZAP-70
binding to the TCR blocks the tyrosine phosphorylation and activation
of ZAP-70 and results in reduced tyrosine phosphorylation of cellular
substrates.
The ability to target and disrupt specific phosphoprotein-SH2 domain interactions holds tremendous potential in the development of reagents to aid in further analysis of signaling pathways and, perhaps more importantly, in the development of rationally designed chemotherapeutic agents, which could see application as novel immunosuppressive or antineoplastic drugs(43) . These studies represent a significant step toward this goal and demonstrate that synthetic PTPase-resistant phosphotyrosyl analog-containing peptides, based on SH2 domain binding sequences, can be successfully introduced into cellular systems to disrupt signaling pathways.
Note Added in Proof-An agreement was reached recently by a group of researchers in the field concerning the nomenclature of the motif referred to in this manuscript as a TAM and which has been alternatively named an ARH1, ARAM, YXXL, or Reth motif. The newly accepted term for this motif is ITAM for Immunoreceptor Tyrosine-based Activation Motif (submitted for publication).