(Received for publication, March 1, 1995; and in revised form, June 1, 1995)
From the
Signaling by the T-cell antigen receptor (TCR) involves both
phospholipase C (PLC)-
Ligation of the T cell antigen receptor (TCR) ( Although the role of PTKs
in the TCR-activated Ras pathway still needs to be clarified, studies
on receptor tyrosine protein kinases (RTK) have shown that a guanine
nucleotide exchange factor (GNEF), human son-of-sevenless (hSos), is
involved(22, 23) . hSos activates p21 To understand the involvement of
Grb2 in TCR-mediated signaling, we wished to identify the
phosphoproteins in the Grb2 complex, including the presence of
PLC-
Figure 1:
TCR
ligation induces the selective association of Grb2 with specific
tyrosine phosphoproteins. Jurkat cells (2
We compared the
profile of Grb2-associated phosphoproteins in the immune precipitates
with phosphoproteins exhibiting binding affinity for a GST-Grb2 fusion
protein. Cellular lysates were incubated with GST-Grb2 immobilized on
glutathione-agarose beads, and the adsorbed proteins were analyzed on
the same anti-phosphotyrosine immunoblot (Fig. 1). In lysates
from anti-CD3-treated cells, GST-Grb2 complexed with p145, p110, p95,
p60-70, and p54 (Fig. 1, lanes8 and 9). To study the contribution of SH2 and SH3 domains of Grb2
in these interactions, we conducted parallel affinity binding
experiments with GST-Grb2(49L/203R), in which both the amino- and
COOH-terminal SH3 domains have been mutagenized to render them
non-functional, while the SH2 domain remains intact(25) .
Interaction of the mutant fusion protein with lysates from
anti-CD3-treated cells showed strong affinity for p36/p38 as well as
additional tyrosine phosphoproteins in the range of 70-75 kDa (Fig. 1, lanes12 and 13). PMA
treatment did not induce the binding of any tyrosine phosphoproteins
with either GST-Grb2 or GST-Grb2(49L/203R) (Fig. 1, lanes10 and 14). Taken together, these results
suggest that both the SH2 as well as the SH3 domains of Grb2
participate in binding of anti-CD3-induced tyrosine phosphoproteins.
Figure 2:
TCR
ligation induces Grb2 association with hSos1. A, anti-hSos1
immunoblot. Jurkat cells (2
When hSos1 immunoprecipitates from
anti-CD3-treated cells were probed by anti-phosphotyrosine
immunoblotting, we observed that these precipitates included a major
36-38-kDa phosphoprotein as well as phosphoproteins of 54, 70,
and 95 kDa (Fig. 3A, lanes3, 4, 6, 7). Taken together with the results in Fig. 2, this finding is compatible with a CD3-induced assembly
of Grb2, hSos, p36/p38, p70, p54, and p95.
Figure 3:
Anti-hSos1 immunoprecipitates (IP) phosphoproteins of the same molecular weight as
anti-Grb2, but Shc does not coprecipitate with Grb2. A,
anti-phosphotyrosine immunoblot of anti-hSos1 immunoprecipitate. Jurkat
cells (2
The 54-kDa phosphoprotein
(p54) shown in lanes3, 4, 6. and 7 (Fig. 3A) is of the approximate size of
p52 The 95-kDa phosphoprotein in lanes3, 4, 6, and 7 is the same size as
p95
Figure 4:
PLC-
Since PLC-
Figure 5:
ZAP-70
associates with the PLC
To confirm that
ZAP-70 associates with the pool of Grb2, which is complexed to
PLC-
Figure 6:
Grb2,
ZAP-70, and PLC-
To
study the intracellular distribution of p36/p38, the cytosolic as well
as Nonidet P-40-soluble and -insoluble fractions were subjected to
anti-Grb2 immunoprecipitation followed by anti-phosphotyrosine
immunoblotting (Fig. 6B). While anti-CD3 induced
association of p110, p75, p70, p54, p21, and p18 with Grb2 in Nonidet
P-40-soluble and -insoluble fractions, there was an interesting
difference in p36/p38 association with Grb2 in these fractions (Fig. 6B, lanes4-9). p36/p38
was inducibly associated with Grb2 in Nonidet P-40-soluble fractions (Fig. 6B, lanes4-6) but was
constitutively associated with Grb2 in Nonidet P-40-insoluble extracts (lanes7-9). The reason for the constitutive
phosphorylation and association of p36/p38 with Grb2 in
detergent-insoluble material is not known. Identical results to that
shown in Fig. 6B were obtained when cytosol and
detergent-soluble and -insoluble fractions were affinity interacted
with PLC- The p18
and p21 phosphoproteins seen at the bottom of Fig. 6B are of the same molecular weight as the TCR- In this paper, we show that Grb2 plays a role in signaling
via the TCR by recruiting hSos1 to a signaling complex that includes
PLC- Grb2 plays a critical
role in signaling by RTKs and receptor-associated
PTKs(25, 26) . Previous studies have highlighted the
potential importance of Grb2 in signaling by the
TCR(20, 21, 28, 32) . Sieh et
al.(20) demonstrated a prominent p36/p38 phosphoprotein
that associated with the SH2 domain of Grb2 and formed a stable complex
with Grb2, hSos1, and PLC- Gauged from the
number of proteins that are complexed to Grb2 (Fig. 1), it is
quite plausible that Grb2 plays an additional role in TCR signaling. Of
particular interest is the presence of PLC- The presence of ZAP-70 in the
Grb2-associated complex and its redistribution to the non-cytosolic
cellular compartment are likely of considerable importance both in the
formation of the complex as well as for ZAP-70 function. ZAP-70 can
associate with one or more of the ARH motifs present in the CD3 complex
( Another
interesting finding of this study is the redistribution of
Grb2-associated components from the cytosol to detergent-soluble and
-insoluble fractions upon anti-CD3 treatment (Fig. 6A).
Utilizing a detergent-soluble fraction, Sieh et al.(20) and Buday et al.(21) have shown
that p36/p38 has a predilection for the particulate fraction of the
cell. However, these authors did not investigate the
detergent-insoluble fraction, which appears to show changes equally
dramatic to that of the detergent-soluble fraction (Fig. 6A). Although it remains to be proven, the
relatively high spectrin content in the detergent-insoluble fraction
suggests that this cell fraction is enriched for select cytoskeletal
proteins (Fig. 6A). It is possible that many of the
TCR-associated signaling events take place in the interface between the
cell membrane and the cytoskeleton. To this end, it is interesting that
among the TCR subunits, the Although Sieh et
al.(20) detected low levels of tyrosine phosphorylation
of Shc in anti-CD3-treated Jurkat cells, in a previous study we failed
to detect similar phosphorylation. Moreover, in this study we found no
evidence for TCR-induced Shc/Grb2 association in four different T-cell
types tested (Fig. 3B). The results in T-cells were in
direct contrast with prominent Shc/Grb2 association in a B-cell line,
Ramos, during antigen receptor ligation (Fig. 3B).
Although this could indicate that Shc does not play an important role
in TCR signaling, Ravichandran et al.(48) showed Shc
binding to TCR-
1 and p21
activation.
While failing to induce Shc/Grb2 association, ligation of the TCR/CD3
receptor in Jurkat T-cells induced hSos1-Grb2 complexes. In addition to
hSos1, Grb2 participates in the formation of a tyrosine phosphoprotein
complex that includes 145-, 95-, 70-, 54-, and 36-38-kDa
proteins. p145 was identified as PLC-
1 and p70 as the protein
tyrosine kinase, ZAP-70. Although of the same molecular weight, p95 was
not recognized by an antiserum to p95 Vav. The SH2 domains of Grb2 and
PLC-
1 were required for the formation of this protein complex. In
anti-CD3-treated cells, Grb2 redistributed from the cytosol to a
particulate cell compartment along with p36/p38, ZAP-70, and
PLC-
1. Part of the Grb2 complex associated with the particulate
compartment could be extracted with Nonidet P-40, while the rest was
Nonidet P-40 insoluble. In both the detergent-soluble and -insoluble
fractions, Grb2 coimmunoprecipitated with the
-chain of the TCR.
Taken together, these results indicate that anti-CD3 induces
Grb2-hSos1-PLC-
1-p36/p38-ZAP70 complexes, which localize in the
vicinity of TCR-
.
)initiates signals at the cell membrane that control
nuclear events such as induction of immediate early response genes and
cytokine gene expression(1) . Two examples of
membrane-to-nuclear signaling are the Ras/mitogen-activated protein
kinase/c-Jun and Ca
/calcineurin/NF-AT cascades, which
both exert control over transcriptional initiation of the interleukin-2
gene(2, 3, 4, 5, 6, 7, 8, 9) .
Both pathways, in turn, are dependent on a TCR-associated protein
tyrosine kinase (PTK) network, which minimally include 3 PTKs:
p56
, p59
, and
ZAP-70(10, 11, 12, 13, 14) .
The link between [Ca
]
flux and PTK activity is the activation of phospholipase
C-
1 (PLC-
1) by TCR-induced tyrosine
phosphorylation(15, 16) . PLC-
1 initiates the
breakdown of inositol phospholipids, thereby generating inositol
trisphosphate, which acts as an intracellular Ca
ionophore. PLC-
1 activation is impaired in an Lck-deficient
Jurkat cell line, suggesting a kinase-substrate
interaction(17, 18) . Moreover, PCL-
1 has been
shown to coprecipitate with an unidentified 36-38-kDa tyrosine
phosphoprotein (p36/p38), which is responsive to TCR signaling (19, 20, 21) .
by the exchange of GDP for GTP(22, 23) .
hSos is linked to RTKs through the interposition of the adaptor
proteins, Shc and Grb2 (24, 25) . Grb2 is comprised
wholly of an SH2 domain flanked by two SH3 domains(26) . The
SH2 domain of Grb2 binds tightly to phosphotyrosine residues on RTKs or
an interposed Shc molecule(25) . Shc, in turn, recognizes
tyrosine-phosphorylated domains on RTKs via its own SH2
domain(25, 27) . Grb2 binding to Shc requires Shc to
be tyrosine phosphorylated by the RTK or a receptor-associated
PTK(25, 27) . By way of one of its SH3 domains, Grb2
binds and recruits hSos to the cell membrane(25, 28) .
In addition to hSos, T-cells contain another putative GNEF, Vav, but
these data are still controversial(29) . While we and others
have previously shown that TCR ligation induces tyrosine
phosphorylation of p95
in Jurkat cells without
inducing Shc/Grb2 complexes, there is growing evidence for involvement
of hSos in TCR
signaling(2, 20, 21, 23, 30, 31) .
It is possible, therefore, that Grb2 may be involved by binding to a
receptor-associated tyrosine phosphoprotein other than Shc. To this
end, our own preliminary data as well as published data from other
groups have shown that Grb2 associates with several tyrosine
phosphoproteins upon TCR
ligation(20, 21, 28, 32) . Included
among these is p36/p38, which is likely identical to the protein that
binds to PLC-
1(20, 21) . These findings suggest
that Grb2 play a role in the assembly of a TCR-associated signaling
complex, which serves to initiate both the inositol phospholipid as
well as the Ras signaling pathways.
1. In addition, we were interested in determining the
localization of this complex with respect to the TCR. We show here that
anti-CD3 stimulation of Jurkat cells induces
Grb2-hSos1-PLC-
1-p36/p38-ZAP-70 complexes, which do not include
Vav or Shc. Grb2, which is predominantly a cytosolic protein, could be
seen to redistribute to both the detergent-soluble and
detergent-insoluble cellular fractions during TCR ligation. Moreover,
in both fractions Grb2 coimmunoprecipitated with the
-chain of the
TCR(33, 34) . These results imply that Grb2 is
intimately involved in TCR signaling and participates in formation of a
TCR-associated signal complex, which induces a convergence of
components linked to multiple signaling pathways.
Antisera and Reagents
The monoclonal
antibody (mAb) OKT3 (anti-CD3) was from Ortho Pharmaceuticals (Raritan,
NJ). Biotinylated goat-anti-human IgM was purchased from Southern
Biotechnology Associates (Birmingham, AL). The anti-phosphotyrosine
mAb, 4G10, was from Upstate Biotechnology Inc. (Lake Placid, NY). Other
mAbs used in this study included anti-Grb2 from Transduction
Laboratories (Lexington, KY) and anti-PLC-1 from Upstate
Biotechnology Inc. The polyclonal antisera to ZAP-70 (antiserum 833)
was previously described(35) . The polyclonal anti-
-chain
antibody, N-40, was kindly provided by Dr. C. Terhorst (Harvard Medical
School, Boston, MA)(30) , while a monoclonal
anti-
-antibody was kindly provided by Dr. R. Kubo (Cytel Corp.,
San Diego, CA)(36) . Polyclonal anti-Grb2 used for
immunoprecipitation studies and anti-hSOS1 were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA), while anti-Shc was obtained from
Upstate Biotechnology Inc. A polyclonal spectrin antiserum was
purchased from Sigma. The PLC-
1 SH2-Ig fusion protein was
previously described(19) .
Bacterially Produced Fusion Proteins
The
cDNA encoding GST-Grb2 Myc (wild type) and PSVEGrb2 Myc49L/203R,
carrying a proline to lysine substitution at residue 49 in the
amino-terminal SH3 domain and a glycine to arginine substitution at
residue 203 in the COOH-terminal SH3 domain of Grb2(25) , were
generously provided by Dr. R. A. Weinberg (Whitehead Institute,
Cambridge, MA). The BamHI-XbaI fragment containing
Grb2 myc49L/203R in the latter plasmid was subcloned into BamHI-XbaI-digested PGEX-20T vector as described
earlier(25) . Both fusion proteins were purified by binding to
glutathione-agarose and eluted with reduced glutathione.Cell Culture, Stimulation, and Lysis
All
cell lines were grown in RPMI 1640 medium supplemented with 10% fetal
calf serum and antibiotics (complete medium). The cell lines used in
this study included the Jurkat clone, 6.1 E (CD2,
CD3
, CD4
), and Ramos cells, a human
mIgM
B cell line established from a patient with
Burkitt's lymphoma. In all experiments, aliquots of 2
10
cells were used unless otherwise stated. All
stimulations were conducted in a total of 1 ml of complete medium.
Jurkat cells were treated with 1 µg/ml OKT3 (anti-CD3) or 100
nM PMA for the indicated time periods at 37 °C. Ramos
cells were preincubated with 2 µg/ml goat-anti-human IgM for 2 min
at 37 °C and then treated with 50 µg/ml avidin for 1-5
min. Cell pellets (12,000
g) were lysed in 0.2 ml of
an appropriate lysis buffer (see below) by sonication. Particulates
were removed at 12,000
g, and the supernatants were
used for immunoprecipitation and Western blotting.
Immunoprecipitation
For Grb2, hSos1, and
ZAP-70 immunoprecipitations, cells were lysed in buffer A (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 10 mM NaF, 2 mM PMSF, 10
µg/ml leupeptin, and 0.009 TIU/ml aprotinin), and the lysates were
incubated with l µg of anti-Grb2, 1 µg of anti-hSos1, or 5
µl of anti-ZAP-70 for 1 h at 4 °C. Thereupon, 30 µl of
protein A-Sepharose beads were added for 1 h at 4 °C. The
immunoprecipitates were washed three times with 0.5 ml of buffer B (20
mM Tris, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 1
mM sodium vanadate, 5 mM NaF, 2 mM PMSF, 10
µg/ml leupeptin, and 0.009 TIU/ml aprotinin) and then boiled in 60
µl of 1 SDS sample buffer for 5 min. The proteins were
resolved by 10% SDS-PAGE and subsequently transferred to Immobilon-P.
For
-chain immunoprecipitation, cell extracts were precleared and
incubated with 10 µl of polyclonal N-40 antiserum for 1 h at 4
°C, followed by incubation with 30 µl of protein A-Sepharose
beads for 1 h at 4 °C(37) . The immunoprecipitates were
washed three times with 0.5 ml of buffer C (20 mM Hepes, pH
7.5, 150 mM NaCl, 0.1% Nonidet P-40, 0.1 mM sodium
vanadate, 0.1 mM PMSF, 1 µg/ml leupeptin, 0.009 TIU/ml
aprotinin) and boiled in 60 µl of 1
SDS sample buffer. The
immunoprecipitates were resolved by 15% SDS-PAGE under reducing
conditions and subsequently transferred to Immobilon-P.
GST-Grb2 and GST-Grb249L/203R Affinity
Association
Cells were lysed in 0.2 ml of buffer D (20
mM Tris, pH 7.4, 1% Triton X-100, 50 mM NaCl, 1
mM sodium vanadate, 10 mM sodium pyrophosphate, 5
mM EGTA, 0.1% bovine serum albumin, 10 µg/ml leupeptin, 10
mM NaF, 0.009 TIU/ml aprotinin). Supernatants were incubated
with 10 µg each of recombinant GST, GST-Grb2 Myc or GST-Grb2
Myc49L/203R and added to 30 µl of glutathione-agarose beads for 1 h
at 4 °C(25) . The beads were washed three times with 0.5 ml
of buffer D lacking bovine serum albumin and boiled in 60 µl of 1
SDS sample buffer. Proteins were resolved by 10% SDS-PAGE and
transferred to Immobilon-P.
PLC-
Lysates were prepared in 0.2 ml of buffer A
containing 5 mM dithiothreitol and incubated with 50 µl of
protein A-Sepharose beads precoated with 30 µg of PLC-1 SH2/Ig Affinity
Association
1 SH2-Ig
fusion protein for 1 h at 4 °C(19) . The beads were washed
three times with 0.5 ml of buffer B containing 5 mM
dithiothreitol and then boiled in 60 µl of 1
SDS sample
buffer. The proteins were resolved by 10% SDS-PAGE and transferred to
Immobilon-P.
Subcellular Fractionation
The pellet of
10 cells was resuspended in 1 ml of chilled hypotonic
buffer E (10 mM Tris, pH 7.4, 10 mM KCl, 1.5
mM MgCl
, 0.2 mM PMSF, 1 mM sodium vanadate, 10 µg/ml leupeptin and 0.009 TIU/ml
aprotinin) for 15 min and then lysed in a Dounce homogenizer by 50
mechanical strokes. All subsequent steps were performed at 4 °C.
Lysates were spun for 30 min at 100,000
g in a Beckman
TL-100 table top centrifuge, and the supernatant, designated cytosol,
was collected. The pellet was rinsed with 2 ml of buffer E,
recentrifuged at 100,000
g, and subsequently
resuspended in 0.2 ml of buffer E plus 1% Nonidet P-40 for 15 min.
Following recentrifugation at 100,000
g for 30 min,
the supernatant, designated detergent-soluble material, was recovered.
The pellet was washed with 0.2 ml of buffer E containing Nonidet P-40
and recentrifuged for 10 min at 100,000
g. The pellet
was solubilized by sonication in 0.2 ml of RIPA buffer (20 mM Tris, pH 8.0, 0.15 M NaCl, 0.1% SDS, 1% Nonidet P-40,
0.5% deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 10 mM NaF, 1 mM PMSF, 10
µg/ml leupeptin, and 0.009 TIU/ml aprotinin) and centrifuged for 30
min at 100,000
g. The supernatant, designated
detergent-insoluble material, was recovered. We confirmed by enzymatic
assay that the detergent-soluble and detergent-insoluble fractions were
substantially free from lactate dehydrogenase activity.
Western Blotting
SDS-PAGE gels were
transferred to Immobilon-P at 0.35 mA for 16 h. The membrane was
blocked with 6% bovine serum albumin, pH 7.6, for 2 h and incubated
with the desired antibody, diluted in blocking solution plus 0.1%
Tween-20 and 0.01% azide for 1 h at room temperature(2) .
Detection was performed either by using the enhanced chemiluminescence
(ECL) method or by overlaying the blot with I-protein A
(1 µCi/ml) in blocking solution for 1 h, followed by four 10-min
washes in Tris-buffered saline containing 0.1% Tween 20 and 0.1% bovine
serum albumin(2) . The blots were visualized by
autoradiography.
TCR Ligation Induces the Association of Grb2 with a
Select Range of Tyrosine Phosphoproteins
Jurkat cells,
either untreated or stimulated with anti-CD3 mAb, were lysed, and
anti-Grb2 immunoprecipitates were prepared. These proteins were
transferred to Immobilon-P and immunoblotted with anti-phosphotyrosine
mAb. Fig. 1shows that anti-CD3 stimulation induced the
association of Grb2 with a number of tyrosine phosphoproteins,
including p110, p95, p75, p60-p70, p54, as well as a p36/p38 doublet (lanes4 and 5). Longer exposure of the blot
also revealed other less prominent phosphoproteins, including p145.
P36/p38 was consistently the most prominent tyrosine phosphoprotein in
the immunoprecipitate. In contrast to ligation of the CD3 receptor, PMA
treatment did not induce complexing of any of the above tyrosine
phosphoproteins with Grb2 (Fig. 1, lane6).
Similar findings were made upon anti-CD3 stimulation of additional
T-cell lines, HPB-ALL and Hut-78 (not shown).
10
) were
left untreated or were treated with 1 µg/ml OKT3 for 1 or 5 min or
with PMA for 5 min and then lysed. Lysates, representing
detergent-soluble protein, were incubated with 1 µg of polyclonal
anti-Grb2 antibody immobilized on protein A-Sepharose or with 10 µg
of either GST-Grb2 myc or GST-Grb2 Myc49L/203R bound to
glutathione-agarose beads as indicated. Precipitation with normal
rabbit serum (NIS) or GST bound to glutathione beads was used
as a negative control. Proteins bound by the antibody or fusion protein
were resolved by 10% SDS-PAGE and electrophoretically transferred to an
Immobilon-P membrane. The membrane was overlaid with 1 µg/ml
anti-phosphotyrosine mAb (4G10) and developed by
ECL.
TCR Ligation Induces Grb2 Association with hSos1
without Involving Shc
Since hSos1 is not a tyrosine
phosphoprotein, we determined whether this protein can coprecipitate
with Grb2 as part of the Grb2 complex. First, we showed that anti-Grb2
immunoprecipitates contain immune detectable hSos1 (Fig. 2A). hSos1/Grb2 association was extremely rapid,
and hSos1 could be visualized within 30 s of CD3 receptor engagement (Fig. 2A, lanes3-8).
Reciprocally, we demonstrated that anti-hSos1 coimmunoprecipitated Grb2
in anti-CD3-treated but not unstimulated Jurkat cells (Fig. 2B). In an anti-hSos1 immunoblot of a parallel
immunoprecipitate (Fig. 2B, bottom), we showed
that equal amounts of hSos1 protein were being precipitated from
control (lane2) and stimulated cells (lanes3 and 4).
10
) were untreated
(Ø) or treated with 1 µg/ml OKT3 for the indicated time
periods. Lysates were subjected to immunoprecipitation using 1 µg
of polyclonal anti-Grb2 antibody. NIS and 100 µg of crude Jurkat
cell lysate were included as controls. The immunoprecipitates (IP) were resolved by 10% SDS-PAGE. After transfer to
Immobilon-P, the membrane was sequentially overlaid with 1 µg/ml
polyclonal anti-hSos1 and 50 ng/ml horseradish peroxidase-conjugated
goat anti-rabbit secondary antibody. The blot was developed by ECL. B, anti-Grb2 and anti-hSos1 immunoblot of an anti-hSos1
immunoprecipitate. Stimulated or unstimulated cell lysates were
subjected to immunoprecipitation using 1 µg of polyclonal
anti-hSos1 antibody. NIS and 100 µg of crude lysate were included
as controls. The proteins were resolved by 10% SDS-PAGE and transferred
to Immobilon-P. The blot was overlaid with 1 µg/ml anti-Grb2 mAb
and then developed by ECL. The 185-kDa region of the blot is shown. The
blot was stripped and overlaid with 1 µg/ml anti-hSos1 polyclonal
antibody (bottom). Lanes1-4 are the
same as lanes1-4 in the upperpanel.
10
) were left untreated (Ø) or
treated with 1 µg/ml OKT3 for 1 or 5 min and then lysed. Lysates
were immunoprecipitated with two different types of anti-hSos
antibody (Ab 1, UBI; Ab 2, Santa Cruz Biotechnology) as described
in Fig. 2A. NIS was included as control.
Immunoprecipitates were resolved by 10% SDS-PAGE. Anti-phosphotyrosine
immunoblotting was performed as in Fig. 1. B, anti-Shc
immunoblot of Grb2 immunoprecipitates. Jurkat cells (2
10
) were either left untreated (Ø) or treated with 1
µg/ml OKT3 for 1 or 5 min or 100 nM PMA for 5 min. Ramos
cells (2
10
) were either left untreated (Ø)
or treated with 2 µg/ml biotinylated goat anti-human IgM + 50
µg/ml avidin for 1 or 5 min or 100 nM PMA for 5 min.
Lysates were immunoprecipitated with anti-Grb2 antibody as described in Fig. 1. NIS and crude lysates served as controls. Proteins were
resolved by 10% SDS-PAGE and transferred to Immobilon-P. The blot was
developed with 1 µg/ml polyclonal anti-Shc antibody, followed by
0.1 µCi/ml
I-protein A. The 40-66-kDa region of
the autoradiogram is shown.
(24, 25, 26) .
Immunoblotting for Shc failed, however, to identify p54 as Shc (not
shown). Moreover, anti-Grb2 antibodies did not coprecipitate Shc in
anti-CD3-treated Jurkat cells (Fig. 3B, lanes2-5). By contrast, cross-linking of the antigen
receptor (mIgM) of a human B-cell line, Ramos, with anti-IgM showed
receptor-induced association of p52
with Grb2 (Fig. 3B, lanes9 and 10).
Among T-cells, these findings were not unique for the Jurkat cell line,
as we have also been unable to show Shc complexing to Grb2 in HPB-ALL,
Hut-78, and peripheral blood PHA blasts during CD3 ligation (not
shown).
, which undergoes enhanced tyrosine
phosphorylation during anti-CD3 treatment in Jurkat
cells(2, 29) . Since Vav is a putative GNEF in
T-cells(29) , its coprecipitation with a definitive GNEF,
hSos1, may explain this controversial aspect of Vav function (38) . Overlay of the blot in Fig. 3A failed to
identify p95 as Vav (not shown). Moreover, we have previously shown
that Vav immunocomplexes do not include p36/p38 (2) or Grb2
(not shown). Although anti-Vav leads to coprecipitation of a 70-kDa
tyrosine phosphoprotein(2) , this protein does not share immune
identity with ZAP-70 (not shown) and is likely the so-called
Vav-associated protein (2, 39) . Taken together, these
findings exclude participation of Vav in Grb2-hSos1 interactions but
do not exclude Vav participation in TCR signaling.
PLC-
Previous reports have shown that TCR
ligation induces tyrosine phosphorylation of PLC-1 Interacts with the Grb2 and p36/p38 via
Its SH2 Domain
1, which
coprecipitates with a 36-38-kDa tyrosine
phosphoprotein(19, 20) . Parallel immunoprecipitation
of PLC-
1 and Grb2, followed by anti-phosphotyrosine
immunoblotting, demonstrated complexing of p36/p38 to PLC-
1 as
well as Grb2 (Fig. 4A). This is in agreement with what
we and others have shown during TCR ligation (19, 20, 21) . Moreover, in anti-CD3-treated
cells, PLC-
1 and Grb2 coprecipitated with p110, p95, p75, p70, and
p54 (Fig. 4A, lanes3 and 5). Anti-PLC-
1 immunoblotting confirmed that p145 (Fig. 4A, lanes2 and 4) is
identical to PLC-
1 (not shown). In a separate experiment, Grb2
immunoprecipitates were analyzed by anti-PLC-
1 immunoblotting. Fig. 4B shows that anti-CD3 treatment induced rapid
(<30 s) association of PLC-
1 with Grb2 (lane3) and that the association was sustained for at least 30
min (lane8).
1 associates with Grb2 complexes
via its SH2 domain. A, anti-phosphotyrosine immunoblot of
PLC-
1 and Grb2 immunoprecipitates. Jurkat cells (2
10
) were left untreated (Ø) or treated with 1
µg/ml OKT3 for 1 min or 5 min and then lysed. Lysates were
immunoprecipitated with 5 µl of anti-PLC-
1 antiserum or 1
µg of polyclonal anti-Grb2 antibody. NIS was included as control.
The immunoprecipitates were resolved by 10% SDS-PAGE, and
anti-phosphotyrosine immunoblotting was done as in Fig. 1. This
blot was overexposed relative to that in Fig. 1. B,
anti-PLC-
1 immunoblot of a Grb2 immunoprecipitate. Anti-Grb2
immunoprecipitation was performed as in Fig. 1. NIS and crude
lysate were included as controls. Proteins were resolved by 10%
SDS-PAGE. After transfer to Immobilon-P, the membrane was overlaid with
1 µg/ml anti-PLC-
1 mAb. The blot was developed by ECL. The
185-110-kDa region of the autoradiogram is shown. C,
anti-phosphotyrosine and anti-Grb2 immunoblot of PLC-
1
SH2/Ig-binding proteins. Lysates were incubated together with protein
A-Sepharose beads coated with 30 µg of PLC
1 SH2-Ig fusion
protein in the presence of 5 mM dithiothreitol. Beads were
extensively washed and boiled in 1
SDS sample buffer. Proteins
were resolved by 10% SDS-PAGE and transferred to Immobilon-P, and
anti-phophotyrosine immunoblotting was performed as in Fig. 1.
For anti-Grb2 immunoblotting, duplicate samples were resolved by 12%
SDS-PAGE, and immunoblotting for Grb2 was performed as in Fig. 1. Lanes1-4 (bottom) are the same
as lanes1-4 in the anti-PY
blot.
1 is a tyrosine
phosphoprotein, its association with Grb2 may involve the SH2 domain of
Grb2. Alternatively, PLC-
1 may use its SH2 domains to bind a
common tyrosine phosphoprotein, such as p36/p38, to which Grb2 is also
complexed. To discriminate between the possibilities, we looked at
T-cell tyrosine phosphoproteins that had affinity for PLC-
1 SH2-Ig
fusion protein. This fusion protein, which contains both the amino- and
COOH-terminal SH2 domains of PLC-
1 (19) , was immobilized
on protein A-Sepharose beads and incubated with control and stimulated
cell lysates. Anti-phosphotyrosine immunoblotting of the eluted
proteins from stimulated cell lysates showed binding of p36/p38, p54,
p70, p75, and p110 to the fusion protein (Fig. 4C, lanes3 and 4). No specific phosphoproteins
bound to PLC-
1 SH2/Ig when lysates from untreated cells were used (Fig. 4C, lane1), while some p75
binding was seen with PMA treatment (lane4). An
anti-Grb2 blot of a parallel PLC-
1-SH2 association experiment
showed the presence of Grb2 in anti-CD3-treated (lanes2 and 3) but not control (lane1) or
PMA-treated (lane4) samples (Fig. 4C, bottom). Since the PLC-
1 SH2
fusion protein does not contain phosphotyrosine residues with which
Grb2 can interact, it is likely that Grb2 is bound to p36/p38 or some
other tyrosine phosphoprotein in the complex. Because p36/p38 is the
most prominent phosphoprotein and is associated with PLC-
1 and
Grb2, it is a likely candidate.
ZAP-70 Associates with the
PLC
Since the
anti-phosphotyrosine immunoblots in Figs. 1A, 3A,
4A, and 4C show a p70 substrate, we explored the
possibility that the kinase ZAP-70 may be included in these complexes.
An anti-ZAP-70 immunoblot of Grb2 immunoprecipitates showed the
presence of ZAP-70 in anti-CD3-treated but not in control or
PMA-treated samples (Fig. 5A, lanes3 and 4). Reciprocally, anti-ZAP-70 coprecipitated Grb2 in
anti-CD3-treated but not control or PMA-treated cells (Fig. 5B, lanes6-9). Compared
with the amount of Grb2 protein present in parallel Grb2
immunoprecipitates (Fig. 5B, lanes2-5), we estimated that about 5% of cellular Grb2
was ZAP-70 associated after anti-CD3 treatment.1-Grb2-hSos1-p36/p38 Complex
1-Grb2-hSos1-p36/p38 complex. A,
anti-ZAP-70 immunoblot of a Grb2 immunoprecipitate. Jurkat cells (2
10
) were treated with 1 µg/ml OKT3 or PMA as
described in Fig. 1. Anti-Grb2 immunoprecipitation was done as
in Fig. 1, and proteins were resolved by 10% SDS-PAGE. The
membrane was overlaid with 1:250 dilution of polyclonal
anti-ZAP-70(833) antiserum, followed by 1 µCi/ml
I-protein A and visualized by autoradiography. B, anti-Grb2 immunoblot of Grb2 and ZAP-70 immunoprecipitates.
Lysates were immunoprecipitated with anti-Grb2 as above or with 5
µl of anti-ZAP-70(833) anti-serum. Proteins were resolved by 10%
SDS-PAGE and immunoblotted for Grb2 as described in Fig. 1. The
23-kDa region of the autoradiogram is shown. C,
anti-PLC-
1 immunoblot of Grb2 and anti-ZAP-70 immunoprecipitates.
Grb2 and ZAP-70 immunoprecipitation were performed as above. The
proteins were resolved by 10% SDS-PAGE and anti-PLC-
1
immunoblotting performed as described in Fig. 4B. D, anti-ZAP-70 immunoblot of ZAP-70 immunoprecipitates.
Anti-ZAP-70 immunoprecipitation was performed as in B, and the
blot was overlaid with 1:1000 dilution of anti-ZAP-70 antiserum,
followed by 1 µCi/ml
I-protein
A.
1, we looked for the presence of PLC-
1 in ZAP-70
immunoprecipitates (Fig. 5C). Anti-PLC-
1
immunoblotting showed rapid association of PLC-
1 with ZAP-70 in
anti-CD3-treated cells (Fig. 5C, lanes7 and 8). A parallel anti-Grb2 immunoprecipitate to show
PLC-
1 coprecipitation was included for comparison (Fig. 5C, lanes2-4). This
result is not due to quantitative differences in the amount of ZAP-70
being precipitated because anti-ZAP-70 overlay showed equal amounts of
protein in stimulated and unstimulated samples (Fig. 5D). This blot also shows that ZAP-70 underwent
hypomobility shift in anti-CD3- and PMA-treated cells (Fig. 5D, lanes3-5). In a
separate experiment, we also determined that hSos1 was present in
anti-ZAP-70 immunoprecipitates (not shown). Taken together, these
results indicate that ZAP-70 is a member of the
PLC-
1-Grb2-hSos1-p36/p38 complex, where it may contribute to the
phosphorylation of one or more components.
Grb2 Redistributes to the Detergent (Nonidet
P-40)-soluble and -insoluble Cellular Fractions in Association with
p36/p38, ZAP-70, and the
Grb2
undergoes intracellular translocation in response to complexing of the
TCR(20) . Following treatment with anti-CD3 mAb for 1 and 5
min, Grb2 protein increased 2.1- and 2.9-fold, respectively, in Nonidet
P-40 extracts of the particulate material (i.e. the pellet
remaining after hypotonic lysis and 100,000 -Chain of the TCR
g centrifugation) (Fig. 6A). There was also
increased association of Grb2 (approximately 2- and 3-fold after 1 and
5 min anti-CD3 treatment, respectively) with the particulate material,
which remains after Nonidet P-40 extraction (Fig. 6A).
Grb2 was released from the so-called Nonidet P-40-insoluble fraction by
treatment of the pellet with RIPA buffer. The detergent-soluble and
-insoluble fractions differ insofar as the insoluble fraction includes
an abundance of 220-240-kDa spectrin dimers, while the
detergent-soluble material contained very little spectrin (Fig. 6A). The cytosol included a small amount of
220-kDa spectrin (Fig. 6A). Because spectrin dimers are
predominantly associated with the Nonidet P-40-insoluble fraction, it
suggests that this fraction may be relatively enriched for select
cytoskeletal components compared to the Nonidet P-40-soluble fraction.
Because Grb2 has been shown in Fig. 4and 5 to associate with
ZAP-70 and PLC-
1, we looked for redistribution of these components
upon anti-CD3 treatment. Fig. 6A confirms their
relocation from the cytosol to the Nonidet P-40-soluble and -insoluble
fractions upon cellular activation (Fig. 6A).
1 redistributes to detergent-soluble and
-insoluble cellular compartments where Grb2 is associated with the
-chain of the TCR. A, immunoblot autoradiograms. Jurkat
cells (1
10
) were untreated (Ø) or treated
with 1 µg/ml OKT3 for 1 or 5 min before lysis in 1 ml of hypotonic
buffer. After centrifugation at 100,000
g for 1 h, the
supernatant was collected as cytosol, and the particulate material was
washed twice in hypotonic buffer and extracted in 200 µl of a
buffer containing 1% Nonidet P-40. Nonidet P-40-soluble material was
collected after recentrifugation. The detergent-insoluble pellet was
washed and then homogenized in 200 µl of RIPA buffer. After
centrifugation at 100,000
g, the supernatants,
designated Nonidet P-40-insoluble material, were recovered. Equal
amounts (100 µg) of Nonidet P-40-soluble and -insoluble protein
along with 80 µl of cytosol were resolved by 10 or 12% SDS-PAGE.
Gels were transferred to Immobilon-P and overlaid with antibodies to
spectrin (1:500 dilution), Grb2, ZAP-70, and anti-PLC-
1 as
described under ``Experimental Procedures.'' Immunoblots were
developed by ECL or
I-protein A overlay as described in Fig. 1, 4, and 5. The autoradiographic density of the blotted
proteins were read in a Micro-Tech densitometer, and their relative
density is displayed below each lane (with the
exception of spectrin, which appeared not to change upon stimulation). B, anti-phosphotyrosine immunoblot of Grb2 immunoprecipitates (IP). Anti-Grb2 immunoprecipitation was conducted using 200
µg of cytosol and Nonidet P-40-soluble and -insoluble material, as
described in Fig. 1. Proteins were resolved by 12% SDS-PAGE and
transferred to Immobilon-P, and anti-phosphotyrosine immunoblotting was
done as described in Fig. 1. The same result was obtained in two
repeat experiments and is identical to a result obtained with
PLC-
1 SH2-Ig fusion protein as affinity matrix. C,
anti-
-immunoblot of a Grb2 immunoprecipitate. Anti-Grb2
immunoprecipitation was performed on 100 µl of Nonidet P-40-soluble
and -insoluble material obtained as described above. Proteins were
resolved by non-reduced 12% SDS-PAGE. The blot was overlaid with 1:1000
dilution of polyclonal anti-
-antibody and detected by 1 µCi/ml
I-protein A. The blot was stripped and reprobed with
4G10, which confirmed that this
-species is tyrosine
phosphorylated (not shown). NIS, non-immune serum control
precipitate from the Nonidet P-40-insoluble fraction of unstimulated
cells. D, anti-Grb2 immunoblot of
anti-
-immunoprecipitate. 10 µl of polyclonal
anti-
-antiserum(N-40) were used to immunoprecipitate 100 µl of
detergent-insoluble material extracted with RIPA buffer. Proteins were
resolved on reducing 12% SDS-PAGE. The blot was overlaid with anti-Grb2
and developed as described in Fig. 2.
1 SH2-Ig fusion protein (not shown). Although some
phosphoproteins in the cytosol coprecipitated with Grb2, no
anti-CD3-induced changes in their association occurred (Fig. 6B, lanes1-3).
Immunoblotting of Grb2 immunocomplexes with antisera to ZAP-70 and
PLC-
1 confirmed the association of these molecules with Grb2
localized to the Nonidet P-40-insoluble fraction (not shown). This
confirms that the coordinate redistribution of Grb2, ZAP-70, and
PLC-
1 in Fig. 6A is due to inclusion in a
multi-molecular complex that undergoes intracellular shift.
-species, which
are phosphorylated when TCR is ligated by an agonist peptide (40) . This suggested that Grb2 may be
-associated. This
notion was strengthened by the ability of polyclonal
anti-
-antiserum to precipitate a phosphoprotein complex from
stimulated cells, which include p145, p110, p95, p70, p36/p38, and p21
(not shown). Moreover, immunoblotting for
-chain following
anti-Grb2 immunoprecipitation showed
coprecipitation in both
Nonidet P-40-soluble and -insoluble extracts (Fig. 6C).
While the amount of
associated with Grb2 increased in Nonidet
P-40-insoluble material upon anti-CD3 treatment, this increase was not
seen in the Nonidet P-40-soluble fraction (Fig. 6C).
Reciprocally, we found that
-chain immunoprecipitation leads to
coprecipitation of Grb2 as shown by immunoblotting (Fig. 6D).
1, the PTK ZAP-70, and a 36-38-kDa tyrosine
phosphoprotein. The complex excluded Shc. The association of Grb2 with
this complex is dependent on its SH2 domain. Grb2 redistributed from a
predominant cytosolic location in unstimulated cells to the particulate
compartment in anti-CD3-treated cells. Moreover, Grb2 associated with
-chain of the TCR in this compartment.
1 upon TCR stimulation. Buday et al.(21) also showed that Grb2-SH2 binds p36 and hSos1. Motto et al.(32) and Reif et al.(28) found a 75-kDa phosphoprotein that associates with
the carboxyl-terminal SH3 domain of Grb2 in anti-CD3-treated T-cells.
In addition, Motto et al.(32) also found a 116-kDa
phosphoprotein in anti-CD3-treated cell lysates that binds to the
amino-terminal SH3 domain of Grb2. Our results are in agreement with
these findings and show, in addition, that ZAP-70 and the
-chain
of the TCR also bind to Grb2 ( Fig. 5and 6). Although the
aggregate effects of Grb2 in TCR signaling still require to be
elucidated, an important role for Grb2 in T-cells appears to be the
activation of p21
(20, 28) . To
this end, several groups have shown that Ras plays an important role in
TCR signaling(6, 30, 31) . We have also
recently highlighted the role of a Ras-dependent serine/threonine
kinase, Raf-1, in the mitogen-activated protein kinase cascade in
Jurkat cells(2) . Although a role for GAP proteins has been
suggested, there is a rapidly developing consensus that Ras activation
in T-lymphocytes is dependent on a GNEF that exchanges GDP for
GTP(20, 21, 28, 30, 31, 32) .
T-lymphocytes are unusual in that they express two potential GNEFs,
hSos1 as well as p95
(29) . Although it
was shown that the TCR in Jurkat cells controls tyrosine
phosphorylation of p95
, the data implicating Vav
as a GNEF are controversial (38) . We provide evidence in this
paper that hSos is involved in TCR responses through association with
Grb2 (Fig. 2). Moreover, this reaction is extremely fast (<30
s) and agrees with the rapid kinetics of Ras, Raf-1, MEK-1, and ERK2
activation in Jurkat cells(2, 41) . Although
anti-phosphotyrosine immunoblotting of Grb2 and hSos1
immunoprecipitates ( Fig. 1and Fig. 3A) show a
95-kDa Grb2-associated phosphoprotein, this protein was not recognized
by anti-Vav antisera (not shown). These findings suggest that Vav does
not function as a component of the Grb2 complex that is induced after
TCR ligation. Our findings do not exclude the possibility, however,
that Vav is associated with the complex in low stoichiometric amounts
or involved in TCR signaling in some other way.
1 and p36/p38 in the
Grb2 complex ( Fig. 1and Fig. 4). Similar proteins were
noticed in Grb2 complexes by Sieh et al.(20) . Our
study shows, in addition, that the SH2 domains of PLC-
1 and Grb2
are involved in complexing to p36/p38 (Fig. 4C). This
suggests that Grb2 and PLC-
1 are independently complexed to
p36/p38 or one of the associated phosphoproteins. It is likely that
p36/p38 is a novel adaptor protein that interacts with Grb2 as well as
PLC-
1. Although PLC-
1 activation requires tyrosine
phosphorylation by a TCR-associated PTK, the relationship between
phosphorylation and activation is not simple. Recently, Yang et
al.(42) demonstrated that EGF-induced activation of
PLC-
1 in hepatocytes requires both tyrosine phosphorylation and
association of PLC-
1 with the cytoskeleton. We suggest that the
shift of PLC-
1 to the detergent-insoluble compartment upon CD3
ligation is required for its activation (Fig. 6A).
However, the accurate assessment of PLC-
1 activation and kinetics
has been hindered by the complex regulatory mechanisms that are
involved in its stimulation, including its tyrosine phosphorylation,
its putative interaction with the actin-associated protein,
profilin(43) , and the translocation of PLC-
1 from the
cellular cytoplasm to the plasma membrane(44) . Whatever the
explanation for PLC-
1 activation in T-cells, its inclusion in a
complex that also involves hSos1 strongly suggests that Ras activation
and inositol/phospholipid turnover are regulated in the same
TCR-associated molecular complex.
,
,
) as well as the
-chain of the
TCR(1, 13, 14) . These motifs contain the
amino acid sequence
YXXL-X
-YXXL and are
phosphorylated by the PTK
p56
(1, 13, 14) . The
added tyrosine phosphate groups are recognized by the SH2 domain of
ZAP-70. One scenario, therefore, is that ZAP-70 is an early participant
that assists in the formation of the Grb2-associated complex by
providing the phosphorylation sites that are necessary to recruit Grb2,
p36/p38, and PLC-
1. ZAP-70 may then phosphorylate PLC-
1 or
other specific substrates. Noteworthy for its absence from the lysates
of T-cells obtained from patients suffering from ZAP-70 deficiency is
an inducible p36 substrate present in lysates from normal human
T-cells(45) . It needs to be confirmed, however, that this
phosphoprotein is the same as p36 in Fig. 1or a substrate for
ZAP-70. Although we have been able to show during in vitro kinase assays that the relative abundance of ZAP-70
autophosphorylation (not shown) is in accordance with the actual amount
of ZAP-70 protein in each fraction (Fig. 6B), it is
difficult to determine whether the kinase is actually activated. This
question awaits the identification of ZAP-70 substrates.
-chain in particular has been found to
localize in a detergent-insoluble T-cell fraction, which may include
the cytoskeleton (46) . The participation of the cytoskeleton
could be important toward the activation of PLC-
1 (see above) as
well as the activation of Raf-1(47) .
in an antigen-responsive T-cell clone. While we do
not understand the reason for these differences, we suggest that the
method of TCR ligation and differentiation state of the T-cell may
determine the involvement of Shc. The alternative use of Shc or another
hypothetical adaptor protein such as p36/p38 may determine signaling
specificity of TCR.
1,
phospholipase C-
1; PTK, protein tyrosine kinase; RTK, receptor
tyrosine protein kinases; GNEF, guanine nucleotide exchange factor;
mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; PAGE,
polyacrylamide gel electrophoresis; NIS, non-immune serum; PMA, phorbol
12-myristate 13-acetate; GST, glutathione S-transferase; TIU,
trypsin inhibitory units.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.