From the
Tyrosine-based activation motifs (TAMs) define a conserved
signaling sequence,
EX
T cell activation is controlled by the antigen receptor
(TCR)
The earliest biochemical response elicited by the TCR is the
activation of protein tyrosine kinases
(9) . One essential
protein tyrosine kinase controlled signaling pathway to originate from
the TCR involves the guanine nucleotide-binding proteins
p21ras
(10) . A recent report has described that the adapter
protein Shc, which is a substrate for TCR-activated protein tyrosine
kinases, can bind to the TCR
Transfection of
chimeric receptors where the receptor cytoplasmic tail comprises a
single TAM which contains 2 tyrosine residues is sufficient to activate
TCR signal transduction pathways
(2, 3, 5) .
Initial studies suggested that mutation of either tyrosine within a TAM
would destroy distal signaling functions. The 2 tyrosine residues
within the TAM are necessary for the SH2 domain-mediated association of
the protein tyrosine kinase ZAP-70 with the TCR
complex
(5, 17) . ZAP-70 has two SH2 domains both of
which seem necessary for interaction with the
T cells (2
The data in Fig. 1a show
that Shc binding to 25 µM
To examine further the interactions of
phosphorylated
The SH3 domains of Grb2 bind to effector
proteins that include the Ras exchange protein Sos. Two other Grb2
SH3-binding proteins of 75 and 116 kDa, that are both substrates for
TCR activated protein tyrosine kinases, have also been described in T
cells
(25, 26) . Shc interactions with the TCR
In further experiments to explore whether
The present data show that the phosphorylation requirements
for the binding of the
Recently, the interaction of Shc and the TCR
The
failure of TAM-associated Shc or Grb2 to recruit Sos does not mean that
the Shc and Grb2 binding to the
The present
data confirm that Shc can specifically bind a tyrosine-phosphorylated
TAM and that this binding is inhibited by a tyrosine phosphopeptide Trk
Y490 previously described as a high affinity binding site for the Shc
SH2 domain. Recently, a second phosphotyrosine-binding domain in Shc
has been described that is distinct to the SH2 domain
(30) . The
TAMs were predicted as binding sites for the SH2 domains of
Shc
(31) , and a GST fusion protein of the Shc SH2 domains will
bind to a tyrosine-phosphorylated TAM. However, whether TrkY490 and the
TAM recognize the SH2 domain or the other phosphotyrosine-binding site
on Shc is not yet known. In the present study, competition and peptide
titration experiments indicated that Shc will only bind to micromolar
levels of
We thank Nicola Reilly and Elizabeth Li for the
synthesis and purification of the tyrosine phosphorylated peptides.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
YX
L/IX
YX
L/I, that
couples the T cell antigen receptor to protein tyrosine kinases and
adapter molecules. The present study shows that phosphorylation of both
tyrosines within the motif is required for high affinity binding of the
tyrosine kinase ZAP-70 whereas phosphorylation of the single
COOH-terminal tyrosine within the motif is optimal for the binding of
the adapter Shc. There were also quantitative differences in the ZAP-70
and Shc association with the
1-TAM since nM
concentrations of the doubly phosphorylated
1-TAM are sufficient
for ZAP-70 recruitment whereas micromolar levels of singly
phosphorylated TAMs are necessary for Shc binding. Shc is tyrosine
phosphorylated in antigen receptor-activated T cells and can
potentially form a complex with the adapter molecule Grb2 and could
thus recruit the Ras guanine nucleotide exchange protein Sos into the
antigen receptor complex. The present data show that Grb2 can bind to
the phosphorylated TAM, but this binding is independent of Shc and
there is no formation of
1-TAM
Shc
Grb2
Sos
complexes in antigen receptor-activated cells. Accordingly, Shc
function should not be considered in the context of Grb2/Sos
recruitment to the T cell antigen receptor complex.
(
)
CD3 complex which comprises the
polymorphic TCR
and
subunits, the invariant polypeptide
chains of the CD3 complex (
,
, and
), and the
non-covalently associated
chains. The capacity of the TCR to
transduce signals across the T cell membrane is mediated by the
cytoplasmic domains of these invariant chains of the TCR
complex
(1, 2, 3) . The intracellular tails of
the CD3 and
molecules contain a common motif,
EX
YX
L/IX
YX
L/I, which is
present in a single copy in each of the CD3 chains and triplicated in
the
subunit
(4) . This activation motif is termed the
tyrosine-based activation motif (TAM) and is crucial for TCR coupling
to intracellular protein tyrosine kinases and hence absolutely required
for all subsequent T cell responses
(2, 3, 5) .
TAM motifs are also found in the Ig
and Ig
subunits of the B
cell antigen receptor and in the
and
subunits of the
Fc
RI and Fc
RIIIA receptors (4, 6, 7). As well, two viral
proteins, the gp130 envelope protein of bovine leukemia virus and the
latent membrane protein 2A of Epstein-Barr virus
(8) may use
this evolutionarily conserved signaling motif to activate lymphocytes.
chain (11). By analogy with the role
of Shc in fibroblasts, the coupling of Shc and the
subunit
suggests a possible mechanism for linking the TCR to the Ras guanine
nucleotide binding cycle. Thus T cells express a Ras guanine nucleotide
exchange protein Sos, the homologue of the Drosophila ``son of sevenless'' gene product
(12) . Sos
complexes to the adapter protein Grb2/Sem5
(13) which is
composed of one SH2 domain and two SH3 domains. The SH3 domains of Grb2
bind to Sos whereas the interaction between the Grb2 SH2 domain and
tyrosine phosphorylated molecules such as Shc may regulate the
function-cell localization of
Sos
(13, 14, 15, 16) .
chain (17, 18). The
binding of Shc to the
chain requires TAM phosphorylation, but the
relative importance of phosphorylation of the 2 tyrosine residues
within a TAM for Shc binding have not been determined. Similarly, it
has been described that ZAP-70 binds to nanomolar levels of
phosphorylated TAMs, but similar quantitation of the Shc interaction
with the phosphorylated TAM has not been explored. Accordingly, the
object of the present study was to compare the phosphorylation
requirements for ZAP-70 and Shc binding to the
1-TAM. The ability
of TAM-associated Shc to form a complex with the adapter Grb2 and the
Ras exchange protein Sos was also examined.
Cells, Antibodies, and Synthetic
Oligopeptides
Human peripheral blood-derived T cells were
prepared as described previously (19). The following antibodies were
used in this study: CD3 mAb UCHT1 kindly provided by Prof. Peter
Beverley (University College, London, United Kingdom), Grb2 mAb was
purchased from Affiniti (Nottingham, UK), Sos and Shc polyclonal
antisera and phosphotyrosine (pTyr) mAb 4G10 were purchased from
Upstate Biotechnology Inc., and ZAP-70 polyclonal antisera was
generously given by Dr. Joseph Bolen (Bristol Myers Squibb). Peptides
corresponding to the sequence of the membrane proximal TAM of the human
TCR chain (
1, residues 49-65, QLYNELNLGRREEYDVL) were
synthesized by Nicola O'Reilly (ICRF, London UK), with either
both tyrosines unphosphorylated (
1-YY), both tyrosines
phosphorylated (
1-pYpY) or with a single tyrosine
phosphorylated i.e. residue 51 phosphorylated and residue 62
unphosphorylated (
1-pYY), residue 51 unphosphorylated and
residue 62 phosphorylated (
1-YpY). Control phosphopeptides
corresponded to tyrosine phosphopeptides with defined SH2-domain
binding capacity: EGFR-Y1068 autophosphorylation site of the human EGFR
(PVPEpYINQS); TRK-Y490 the binding site for Shc
(IENPQpYFSDA); PDGFR-Y751-binding site for p85 subunit of
phosphatidylinositol 3`-kinase and Nck (SVDpYVPMLDMK) and
PDGFR-Y1009-binding site for Syp (PVPEpYINQS). GST fusion
proteins of the proline-rich carboxyl-terminal region of Sos (GST Sos)
and wild type Grb2 (GST Grb2) were generated and purified as described
previously
(13, 14) .
10
/ml or as indicated) were either unstimulated or were
activated via the TCR
CD3 complex using the anti-CD3 antibody
UCHT1 (10 µg/ml) or with 20 ng/ml of IL2 for 3 min, or as
indicated, at 37 °C. Cells were pelleted, lysed for 20 min on ice
in 1 ml of immunoprecipitation buffer containing 1% Nonidet P-40, 150
mM NaCl, 50 mM HEPES, pH 7.5, 10 mM
iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1
mM sodium orthovanadate, and 1 mg/ml each of antipain,
chymostatin, leupeptin, and pepstatin and immunoprecipitated as
described previously
(20) . Precipitations using synthetic
peptides were performed using 75 nM-25 µM peptide
as indicated coupled to Affi-Gel 10 affinity matrix (Bio-Rad). Peptides
were coupled according to the manufacturer's recommendations.
Precipitations with GST fusion proteins used 5 µg of fusion protein
immobilized on glutathione-agarose beads.
Western Blotting
Peptide precipitates or
acetone-precipitated protein from whole cell lysates were resolved by
10% SDS-polyacrylamide gel electrophoresis and electroblotted at 0.35
A, 60 V for 5 h onto polyvinylidene difluoride membranes (Millipore,
UK). Filters were blocked overnight at 4 °C with 5% non-fat milk
protein in phosphate-buffered saline, washed in phosphate-buffered
saline, 0.05% Tween 20 and probed with primary antibody as indicated.
Horseradish peroxidase-coupled anti-rabbit or anti-mouse IgG were used
as second stage reagents as required to allow detection by enhanced
chemiluminescence (ECL, Amersham, UK). Quantitation of ECL bands was by
densitometry of autorads as recommended by the manufacturer.
ZAP-70 and Shc Bind to Tyrosine-phosphorylated
In initial experiments we compared the binding of Shc
and ZAP-70 to the tyrosine-phosphorylated 1-TAM
1-TAM. To determine the
relative importance of phosphorylation of the different tyrosine
residues in the protein interactions of TAMs, we used
1-TAM
peptides (see ``Experimental Procedures'') that were
unphosphorylated (
1-YY) or singly phosphorylated on either the
NH
-terminal (
1-pYY) or COOH-terminal
(
1-YpY) tyrosine or phosphorylated on both tyrosines
(
1-pYpY). We selected the
1-TAM for these analyses on
the basis that when expressed as a receptor chimera the
1-TAM is
sufficient for the induction of early and late T cell activation
events
(5) . Equivalent amounts of each of the
1-TAM
peptides coupled to agarose matrix were used to precipitate proteins
from Nonidet P-40 lysates of peripheral blood-derived T cells. Bound
proteins were eluted and resolved by SDS-polyacrylamide gel
electrophoresis, transferred electrophoretically to PVDF membranes, and
subjected to Western blot analysis with anti-ZAP-70 or anti-Shc
antibodies. The data in Fig. 1a show that the doubly
phosphorylated
-TAM peptide precipitates ZAP-70 and Shc from T
cell lysates. ZAP-70 and Shc did not bind to the non-phosphorylated
1-YY peptide or to any of the control tyrosine phosphopeptides
used: the EGFR-Y1068, the PDGFR-Y751, or the PDGFR-Y1009
(Fig. 1a). Densitometry measurements showed that
relative to whole cell lysate the
1-pYpY peptide
precipitated a major fraction of the total cellular ZAP-70
(approximately 70%) and the
1-pYY peptide somewhat less
(approximately 30%), but the
1-YpY peptide was very
inefficient in binding ZAP-70 precipitating approximately 5% of the
total (Fig. 1a). There was no detectable ZAP-70
associated with the non-phosphorylated
1-YY peptide. The most
efficient peptide for Shc binding was the singly phosphorylated
1-YpY peptide (2% of total Shc), the doubly phosphorylated
peptide
1-pYpY was less efficient, and Shc bound very
poorly to the
1-pYY peptide and did not bind to
non-phosphorylated peptide
1-YY (Fig. 1a). Thus,
ZAP-70 and Shc show a preference for binding to different tyrosine
residues when only a single tyrosine within the
1-TAM is
phosphorylated. Anti-Shc immunoblots of total cell lysates show that T
cells express the 46- and 52-kDa isoforms of Shc (Fig. 1). Both
forms of Shc bind to the phosphorylated
1-TAM although there was a
clear preferential binding of the 52-kDa Shc molecule
(Fig. 1a). The
1-pYpY peptides precipitated
a major fraction (approximately 70%) of the total cellular ZAP-70 but
were much less efficient in binding Shc, only precipitating
approximately 1% of the total Shc protein (Fig. 1a). The
optimal TAM peptide for Shc binding,
1-YpY, was also
relatively inefficient for Shc binding, preclearing 2% of total Shc
from cell lysate.
Figure 1:
ZAP-70 and Shc associate with the
1-TAM. T cells (2
10
/point) were lysed, and
proteins were precipitated as follows: a, with 25
µM
1-YY,
1-pYY,
1-YpY,
1-pYpY or
EGFR-Y1068, TRK-Y490, PDGFR-Y751 or PDGFR-Y1009 phosphopeptide or were
acetone-precipitated (Lysate). Lysate in anti-ZAP-70
immunoblot is from 5
10
cells. b, T cells
(2
10
/point) were lysed and precipitated with 25
µM
1-YpY peptide in the presence of increasing
concentrations of TRK-Y490 peptide as shown. Western blots were probed
with anti-ZAP-70 or anti-Shc antibodies as
indicated.
As a positive control for these Shc binding
experiments, the Shc interaction with the -TAM was compared to the
binding of Shc to a tyrosine phosphopeptide, TRK-Y490, which has been
previously described as a high affinity binding site for the Shc SH2
domain
(21) . The data in Fig. 1a show that
TRK-Y490 binds Shc more efficiently than
1-YpY. Hence 25
µM of TRK-Y490 precleared 95% of the Shc from a cell
lysate whereas an equivalent amount of
1-YpY removed only
2% of Shc (Fig. 1a). Moreover, there was a clear
preferential binding of the 52-kDa Shc molecule to
1-TAM peptides
whereas the TRK-Y490 peptide is less discriminatory for the different
isoforms of Shc. The TRK-Y490 peptide did not bind ZAP-70
(Fig. 1a).
1-YpY was completely
inhibited by the TRK-Y490 peptide. TRK-Y490 did not compete ZAP-70
binding to
1-YpY (Fig. 1b). We have also
examined the binding of ZAP-70 and Shc to tyrosine-phosphorylated
peptides corresponding to the two other
-TAMs,
2 and
3.
These peptides also associated efficiently with ZAP-70 but much more
weakly with Shc, exactly analogous to the low efficiency
1-TAM/Shc
association (data not shown).
1-TAM peptides with ZAP-70 and Shc, we precipitated
proteins from T cell lysates with a titration of concentrations of the
TAM peptides (25 µM to 75 nM). The data in
Fig. 2
show titrations of the optimal peptides for ZAP-70-binding
1-pYpY and
1-pYY and the optimal peptide for
Shc-binding
1-YpY. These data (Fig. 2) show that the
doubly phosphorylated
1-pYpY peptide binds ZAP-70 at
concentrations as low as 75 nM. In contrast, equivalent
binding of ZAP-70 to the singly phosphorylated peptide
1-pYY required 1.5 µM of peptide
(Fig. 2b). Thus ZAP-70 binds with approximately a
20-fold higher affinity to a double phosphorylated TAM peptide than to
a single phosphorylated TAM peptide. To analyze the interaction of Shc
with
1-YpY, we precipitated T cell lysates with a titration
of
1-YpY peptide (25 µM to 150 nM)
and Western blotted with anti-Shc antibody (Fig. 2c).
The Shc association with
1-YpY was only detected with
concentrations >7.5 µM of peptide
(Fig. 2c). The differences in the peptide concentrations
required for ZAP-70 versus Shc binding show that the
1-TAM peptides bound Shc with low efficiency compared to
1-TAM/ZAP-70 binding.
Figure 2:
Quantitation of ZAP-70 and Shc binding to
phosphorylated 1-TAM. Proteins from Nonidet P-40 lysates of T
cells (2
10
/point) were precipitated with the
following phosphopeptides coupled to Affi-Gel 10 (a) 15
µM, 1.5 µM, 150 nM or 75 nM
1-pYpY peptide or (b) 15 µM, 1.5
µM, 150 nM, or 75 nM
1-pYY peptide
or (c) 25 µM, 15 µM, 7.5
µM, 1.5 µM, 150 nM
1-YpY
peptide. Western blots were probed with anti-ZAP-70 or anti-Shc
antibodies as indicated.
The effectiveness of the TAMs in affinity
purifying Shc and ZAP-70 from T cell lysates is determined by the
relative affinity of Shc or ZAP-70 for the TAMs and the intracellular
concentrations of the two proteins. Thus the low efficiency of the
1-TAM-Shc interaction compared to the
1-TAM/ZAP-70 binding
could reflect differences in the affinity of Shc and ZAP-70 for the
1-TAM or it could reflect that cellular concentrations of Shc are
lower than ZAP-70 levels. The present data do not resolve these two
possibilities. However, as a comparison we carried out peptide
competition experiments with the TRK-Y490 peptide and the
phosphorylated
1-TAM peptides. These studies indicated that
nM levels of TRK-Y490 were sufficient for Shc binding compared
to the µM
1-YpY peptide concentrations
required for Shc binding (data not shown). The high affinity of
TRK-Y490 for Shc compared to the
1-TAMs is supported by the data
in Fig. 1that show that TRK-Y490 can efficiently preclear
>95% of Shc from T cell lysates whereas the
1-TAMs could purify
a maximum of 1-2% of cellular Shc.
The Adapter Proteins Shc and Grb2 Associate with
Tyrosine-phosphorylated
Shc is tyrosine phosphorylated in TCR-activated T
cells
(11) . Anti-phosphotyrosine Western blots of pTyr
precipitates and acetone precipitates of whole cell lysates showed a
marked increase in tyrosine phosphoproteins following TCR stimulation
(Fig. 3a). Tyrosine-phosphorylated proteins were not
seen in 1-TAM but No
1-TAM
Shc
Grb2
Sos Complexes Are
Detected
1-TAM peptide precipitates from unstimulated cells
(Fig. 3a). ZAP-70 is tyrosine phosphorylated in
TCR-activated cells, and a major tyrosine-phosphorylated 70 kDa band
coprecipitated with the
1-pYpY peptide following
stimulation as well as several weaker bands between 80 and 90 kDa. A
very small amount of the tyrosine-phosphorylated 70 kDa band was also
found to precipitate with the
1-pYY peptide in stimulated
cells (5-10% of that seen with the
1-pYpY peptide).
No 46- or 52-kDa tyrosine phosphoproteins corresponding to
tyrosine-phosphorylated Shc were seen in anti-pTyr Western blots of
1-TAM precipitates from unstimulated or TCR-activated cells
(Fig. 3a). As a positive control, Western blot analysis
of Shc precipitates with anti-pTyr antibodies confirmed the tyrosine
phosphorylation of Shc in TCR-activated cells (data not shown).
Figure 3:
Association of pTyr proteins with the
1-TAM. a, T cells (2
10
/point) were
either unstimulated or stimulated with CD3 mAb UCHT1 (10 µg/ml) for
3 min and were then lysed and proteins precipitated with 25
µM of the following phosphopeptides coupled to Affi-Gel
10:
1-YY,
1-pYY,
1-YpY,
1-pYpY, EGFR-Y1068 as
indicated or pTyr mAb coupled to protein A-Sepharose or were
acetone-precipitated (Lysate, 5
10
cells).
Western blots were probed with anti-pTyr mAb. Molecular mass markers
are in kDa. b, T cells (2
10
/point) were
left unstimulated or were stimulated with CD3 antibody UCHT1 (10
µg/ml) for the times indicated. Cells were lysed, and proteins were
precipitated with GSTGrb2 fusion protein. Acetone-precipitated whole
cell lysates (Lysate, 5
10
cells) were run
in an adjacent lane. Western blots were probed with anti-Shc
antibody.
Tyrosine-phosphorylated Shc has the potential to form a complex with
the adapter protein Grb2
(15, 22) which has been shown
to couple the guanine nucleotide exchange factor Sos to activated
growth factor receptors
(13, 15) . Therefore, we examined
in T cells whether Grb2, Shc, and the 1-TAM are associated. First,
and consistent with the TCR-induced tyrosine phosphorylation of Shc,
the data show that a Grb2 GST fusion protein can bind in vitro to the 52-kDa isoform of Shc in T cell lysates prepared from
TCR-activated but not quiescent T cells (Fig. 3b). The
data (Fig. 4a) show also that Grb2 binds to
tyrosine-phosphorylated
1-TAM peptides interacting preferentially
with a
1-pYpY peptide and binding less efficiently to the
1-pYY peptide. Grb2 did not bind to the
1-YY or
1-YpY peptides. Grb2 binds to the
1-TAM via its SH2
domain as judged by competition experiment where proteins from T cell
lysates were precipitated with
1-pYpY in the presence of
increasing concentrations of a peptide that binds with high affinity to
the Grb2 SH2 domain, EGFR Y1068.
(15, 23, 24) .
The data (Fig. 4a) show a high level of Grb2 binding in
the EGFR-Y1068 peptide precipitates.Fig. 4b shows that
2.5 µM EGFR-Y1068 peptide effectively competed for Grb2
binding to 25 µM of
1-pYpY but did not compete
ZAP-70 binding to
1-pYpY. In Fig. 4c we
precipitated unactivated T cell lysates with a titration of
concentrations of the
1-pYpY peptide coupled to agarose (15
µM to 75 nM). Western blot analyses
(Fig. 4c) showed that Grb2 binding to
1-pYpY was detected only with peptide concentrations higher than 1.5
µM compared to ZAP-70 binding which was readily observed
with 75 nM of peptide (Fig. 4c, cf.Fig. 2a).
Figure 4:
A
comparison of the Grb2/Shc association with the 1-TAM. Anti-Shc,
anti-Grb2, or anti-Sos Western blots of (a) proteins
precipitated from unstimulated or UCHT1-stimulated T cell (2
10
) lysates with 25 µM peptides coupled to
Affi-Gel 10:
1-YY,
1-pYY,
1-YpY,
1-pYpY, EGFR-Y1068
as indicated or acetone-precipitated (Lysate 5
10
cells). b, proteins precipitated from unstimulated T
cell lysates (2
10
/point) with 25 µM
1-pYpY peptide in the presence of increasing concentrations of
EGFR-Y1068 peptide as indicated. c, proteins precipitated from
T cell lysates (2
10
/point) with concentrations of
15 µM, 1.5 µM, 150 nM, or 75
nM
1-pYpY peptide as shown.
These experiments show that Grb2 can
bind to the 1-TAM. However, the ability of the
1-TAM peptides
to bind to Grb2 in lysates of quiescent T cells was apparently not
mediated by Shc as judged by the completely different pattern of
reactivity of these two proteins with the different
1-TAM tyrosine
phosphopeptides (Fig. 4a). In particular Shc bound
preferentially to the
1-YpY peptide which did not bind
Grb2. In TCR-activated cells the pattern of Shc reactivity with the
1-TAM was very similar to the pattern from unactivated cells
(Fig. 4a). Moreover, the reactivity pattern of Grb2 with
the
1-TAM peptides did not change upon T cell activation
(Fig. 4a) whereas the ability of tyrosine phosphorylated
Shc to bind to the Grb2 fusion protein was TCR induced
(Fig. 3b).
chain were proposed as a mechanism to recruit Grb2 and hence the Ras
exchange protein Sos into the TCR complex although Grb2 or Shc/Grb2
could equally recruit p75 or p116 into the TCR complex. The data in
Fig. 3a show that no 75- or 116-kDa tyrosine
phosphoproteins were detected in the
1-TAM precipitates. Moreover,
Western blot analysis failed to detect Sos in the
1-TAM
precipitates isolated from TCR-activated or quiescent T cells
(Fig. 4a). The EGFR-Y1068 peptide can affinity purify
the total cellular pool of Grb2 from T cells and hence the
subpopulation of Grb2
Sos complexes. As a positive control, the
data show that our Western blot analyses could detect Sos in EGFR-Y1068
peptide precipitates.
1-TAM
Shc
Grb2
Sos complexes form, we analyzed the
tyrosine phosphoproteins associated with the SH2 domains of endogenous
Grb2 in TCR-activated cells. It is well established that IL2 induces
formation of Shc
Grb2 complexes in T lymphocytes
(27) .
Therefore, as a positive control, Grb2 complexes from TCR-activated
cells were compared with the Grb2 complexes isolated in parallel from
IL2-activated cells. The data in Fig. 5show that in
IL2-activated T cells a 52-kDa tyrosine phosphoprotein corresponding to
Shc can be detected in the endogenous Grb2 complexes. The identity of
this 52-kDa protein as Shc was confirmed by Western blot analyses with
specific Shc antisera (data not shown). The data in Fig. 5show
also that a TCR-induced 36-kDa tyrosine phosphoprotein is detected in
the endogenous Grb2 complexes, but no 52-kDa tyrosine phosphoprotein
corresponding to Shc was observed. Thus in TCR-activated cells, Shc
binding to GST fusion proteins of Grb2 can be observed
(Fig. 3b), but in vivo Shc
Grb2 complexes
are not detected (Fig. 5). Similarly, no 16-21-kDa tyrosine
phosphoproteins corresponding to the TCR
chain could be detected
in the endogenous Grb2 complexes (Fig. 5). In this context, in
repeated experiments we have failed to demonstrate coprecipitation of
endogenous
chain
Grb2 or
chain
Shc complexes in
peripheral blood T cells (Fig. 5, data not shown).
Figure 5:
A comparison of the TCR- and IL2-induced
Grb2-binding tyrosine phosphoproteins. Antiphosphotyrosine Western
blots of the endogenous Grb2 complexes precipitated from unstimulated,
UCHT1, or IL2-activated T cells (2 10
/point) with
the GST Sos fusion protein. GST Sos binds to the SH3 domains of Grb2
and efficiently purifies the total cellular pool of Grb2 and any
associated Grb2 SH2-binding proteins.
1-TAM to ZAP-70 and Shc were quite
distinct. ZAP-70 bound preferentially to the doubly phosphorylated
motif (
1-pYpY) whereas Shc bound preferentially to a TAM
with a single phosphorylation of the COOH-terminal tyrosine residue
(
1-YpY). ZAP-70 could also bind to a singly phosphorylated
TAM but had a marked preference for a TAM in which the
NH
-terminal tyrosine was phosphorylated
(
1-pYY), binding extremely poorly to the COOH terminally
phosphorylated motif and not at all to the non-phosphorylated TAM. The
presence of the 2 tyrosines within a TAM is proposed as a prerequisite
for optimal ZAP-70
association
(2, 5, 6, 7, 28) .
The present data are in accord with this model since the affinity of
ZAP-70 for the doubly phosphorylated TAM was 20-fold higher than its
estimated affinity for the singly phosphorylated
1-pYY. The
pattern of reactivity of ZAP-70 and Shc with the different phosphoforms
of the
1-TAM highlights the specificity of these interactions as
does the control experiments carried out with a range of tyrosine
phosphopeptides. Interestingly, Shc clearly binds with less efficiency
to the doubly phosphorylated
1-TAM than to the motif containing a
single COOH-terminal phosphotyrosine despite identical sites being
present in both TAMs. This suggests that phosphorylation of the
NH
-terminal tyrosine in the TAM may result in allosteric
effects which interfere with Shc binding to the COOH-terminal tyrosine.
Moreover, the hierarchy of reactivity of ZAP-70 and Shc with the
different phosphoforms of the
1-TAM and the preferential binding
of Shc and ZAP-70 to different phosphotyrosine residues within the
1-TAM may allow the binding of two molecules to a doubly
phosphorylated TAM, thereby facilitating their interaction.
chain was
proposed to couple the TCR to the Ras signaling pathway by recruiting
Grb2
Sos into the TCR complex
(11) . The present data
confirm that Shc is capable of binding to a phosphorylated
1-TAM
but show that this association is relatively inefficient. The best
in vitro binding data showed that only a small subpopulation
of predominantly the 52-kDa isoform Shc (<2%) bound to the
1-TAM, and since no 46- or 52-kDa tyrosine phosphoproteins bound
to the TAM peptides it seems that tyrosine-phosphorylated Shc does not
bind to the TAM peptides. Shc isolated from TCR-activated cell lysates
could bind to a Grb2 GST fusion protein confirming the potential for
Shc and Grb2 to form a complex in T cells. However, TCR-induced
endogenous TAM
Shc
Grb2 complexes were not detected.
Nevertheless, Grb2 could bind to the phosphorylated
1-TAM, but
this association was not influenced by TCR activation. The independence
of Shc/Grb2 association with the
1-TAM is also indicated by the
different preference of the two molecules for different phosphorylated
forms of the
1-TAM. Thus, Grb2 binds best to the doubly
phosphorylated TAM and to the peptide singly phosphorylated on the
NH
-terminal phosphotyrosine whereas Shc bound poorly to the
doubly phosphorylated TAM and of the singly phosphorylated peptides had
a preference for the COOH-terminal phosphotyrosine. Grb2 can bind via
its SH3 domains to the Ras guanine nucleotide exchange protein Sos, but
no Sos was ever detected in
1-TAM precipitates and it is unlikely
that Shc or Grb2 binding to the
chain recruits Sos to the TCR
complex. In particular, although Shc
Grb2 complexes can be readily
detected in IL2-activated T cells they cannot be detected in cell
lysates isolated from TCR-activated cells. Rather, an alternative model
for Sos/p21ras regulation in T cells is suggested by observations that
Sos-associated Grb2 forms a complex predominantly with a 36-kDa
tyrosine phosphoprotein in TCR-activated cells
(26) .
chain is irrelevant because it is
unlikely that the cellular function of Shc and Grb2 is limited solely
to a role in Sos cellular localization-p21ras regulation. For example,
Grb2 SH3 domains do not only bind Sos but are also constitutively
associated with 75- and 116-kDa proteins of unknown
function
(25, 26) . We also failed to detect p75 or p116
in the TAM complexes, but the principal still applies that there are
multiple Grb2 effector complexes in T cells and Grb2 function should
not just be considered in the context of the Grb2
Sos interaction.
Similarly, it has been described that Shc can associate with a 150-kDa
tyrosine phosphoprotein in TCR-activated cells
(29) .
Accordingly, Shc function in T cells should not only be considered in
the context of Grb2
Sos
p21ras regulation.
1-YpY. Grb2 binding to the phosphorylated
1-pYpY peptide was also only observed when micromolar
concentrations of peptides were used compared to the nanomolar range
affinity of ZAP-70 for this doubly phosphorylated TAM. Thus there are
clear quantitative differences between the ZAP-70 and Shc interaction
with the phosphorylated
1-TAM. The relatively high efficiency of
ZAP-70/TAM binding would enable ZAP-70 to form stable complexes with
the tyrosine-phosphorylated
chain whereas the much lower
efficiency of Shc and Grb2 interaction with the TAM may be insufficient
for stable recruitment of these adapters to the
subunits in
endogenous TCR complexes. ZAP70 binding to the TCR is readily
detectable in coprecipitation experiments which is consistent with the
high efficiency of ZAP70/TAM binding shown herein. In contrast, we
found that endogenous TCR
Shc complexes or TCR
Grb2 complexes
were not detectable, but this was also predictable from the current
binding data showing that the Shc and Grb2 interactions with the TAMs
are inefficient. The efficiency of the Shc or Grb2/TAM binding may thus
only allow transient associations that are difficult to maintain during
cell lysis and purification of receptor complexes. Nevertheless, these
interactions might be sufficient in vivo to facilitate
phosphorylation of Shc or Grb2-associated proteins by protein tyrosine
kinases such as ZAP-70 that are stably associated with high affinity
within the TCR complex.
-associated protein
kinase; mAb, monoclonal antibody; GST, glutathione
S-transferase; IL-2, interleukin 2.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.