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(Received for publication, October 6,
1994; and in revised form, November 22, 1994) From the
Products of the crk oncogene are expressed in all
tissues. Crk proteins are composed exclusively of Src homology 2 (SH2)
and Src homology 3 (SH3) domains, and they have been implicated in
intracellular signaling. For example, they participate as mediators of
Ras activation during nerve growth factor stimulation of PC12
pheochromocytoma cells. We examined the role of Crk proteins during T
cell receptor-mediated signaling and observed that Crk proteins
specifically interact, via their SH2 domains, with a
tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116
may be related to the recently cloned fibroblast p130 The product of the v-crk oncogene was identified in the
retroviral genome of avian sarcoma virus-infected
fibroblasts(1, 2) . Three different Crk homologues,
Crk I, Crk II, and Crk-L, have been identified in mammalian
cells(3, 4, 5) . Crk I is composed of a SH2 ( During
v-Crk-mediated transformation of chicken embryonic fibroblasts, three
major proteins of 130, 110, and 70 kDa are tyrosine-phosphorylated and
interact with Crk(1) . While the identity of the 110-kDa
species remains unknown, the 70- and 130-kDa protein have been
identified as the cytoskeletal protein paxillin (14) and the
recently cloned Crk-associated substrate
(p130 We have
examined the role of Crk in T cell activation and report here that,
upon T cell activation, Crk specifically associates via its SH2 domain
with a 116-kDa phosphorylated protein. Either CD4 or CD8 co-receptor
cross-linking to TCR augmented the level of tyrosine phosphorylation of
p116 resulting in its enhanced interaction with Crk. We observed that
glutathione S-transferase (GST)-Crk fusion proteins can
associate with C3G in T cell lysates. Furthermore, in vivo,
Crk-L and perhaps Crk II form a complex with C3G. Thus, Crk, through
its interaction with other proteins via its SH2 and SH3 domains, may
function as an adapter protein in TCR signaling and Ras activation.
The role of the Crk adapter protein in TCR-mediated signaling
was investigated using a murine T cell hybridoma (BYDP). T cells were
activated by antibody-mediated cross-linking of the TCR alone or by
cross-linking the TCR with either the CD4 or CD8 co-receptors. Crk
proteins were immunoprecipitated using an anti-Crk I mAb and
immunoblotted with anti-phosphotyrosine antibody (4G10). Although in
some transformed cells, Crk and Crk-L are tyrosine-phosphorylated (24, 25, 26) , upon T cell activation Crk did
not appear to be tyrosine-phosphorylated; however, a 116-kDa
tyrosine-phosphorylated protein co-precipitated with Crk only upon
activation (Fig. 1A, lane4). p116
did not associate with other SH2/SH3-containing adapter proteins, Nck (Fig. 1A, lanes 5 and 6) or Shc (data
not shown and (27) ). Addition of the Crk immunizing peptide
during Crk immunoprecipitation resulted in the loss of p116 association
(data not shown). Subsequent stripping and reprobing of the
nitrocellulose membrane with the anti-Crk antibody indicated that the
same level of Crk was precipitated in all lanes (Fig. 1B). The anti-Crk mAb used in this study,
although raised against Crk I, cross-reacts with all three isoforms of
Crk (Fig. 1B and data not shown). When Crk II or Crk-L
specific antibodies were used to precipitate individual Crk isoforms,
it was revealed that the p116 protein associates with all three
isoforms of Crk upon TCR-mediated activation (data not shown).
Figure 1:
Crk associates
with p116 upon T cell activation. A, BYDP cells were activated
by antibody-mediated cross-linking, as indicated, for 1 min at 37
°C. Activated (+) or non-activated(-) cells were lysed
with 1% Nonidet P-40, and proteins were immunoprecipitated with
anti-Crk, anti-Nck, or control Ig antibodies and immunoblotted with
anti-phosphotyrosine (Anti-Ptyr) antibody RC20H. B,
immunoprecipitates were also immunoblotted with anti-Crk mAb (lanes3, 4, 9, 10, 13, and 14). The arrows identify different Crk isoforms.
Molecular size markers are shown on the left of all
figures.
It
has been reported that cross-linking CD4 or CD8 with the TCR leads to
enhanced tyrosine phosphorylation of intracellular proteins. Consistent
with this observation, cross-linking of CD4 or CD8 co-receptors with
the TCR resulted in enhanced association of Crk with p116, due to
enhanced phosphorylation of p116 (Fig. 1A, lanes10 and 14). Since both CD4 and CD8 associate
with a Src family tyrosine kinase p56 The kinetics of the Crk-p116 association was assessed in a time
course of T cell activation. T cells were activated by TCR
cross-linking for different times, and Crk proteins were then
immunoprecipitated with anti-Crk mAb and immunoblotted with
anti-phosphotyrosine antibody for the presence of
tyrosine-phosphorylated p116. Phosphorylated p116 associated with Crk
as early as 15 s after activation (Fig. 2, lane3). The level of p116 in Crk immunoprecipitates was
maximal by 3 min after activation, began to decline by 10 min, and
diminished to near-basal levels by 60 min (Fig. 2, lanes
5-7).
Figure 2:
Kinetics of Crk-p116 association. BYDP T
cells were activated by TCR cross-linking for the indicated times and
lysed with 1% Nonidet P-40, and proteins were immunoprecipitated with
anti-Crk mAb (lanes3-14) and immunoblotted
with anti-phosphotyrosine (Anti-Ptyr) antibody
(RC20H).
To determine if Crk-p116 association also occurs in
non-transformed T cells, lysates from human peripheral blood T cells
were immunoprecipitated with anti-Crk mAb and immunoblotted with
anti-phosphotyrosine antibody. As with the T cell hybridoma,
phosphorylated p116 associated with Crk only upon TCR-mediated
activation (Fig. 3, lane 4). There were trace amounts
of p116 associated with Crk in lysates of non-stimulated T cells (Fig. 3, lane 3). This minimal phosphorylation of p116
in resting T cells may reflect heterogeneity in human peripheral T
cells or minimal activation that could occur from the purification
procedure.
Figure 3:
Association between Crk and p116 in
activated human peripheral blood T cells. Purified human T cells were
stimulated by TCR cross-linking with OKT3 for 1 min at 37 °C. Cells
were lysed in 1% Nonidet P-40, immunoprecipitated with anti-Crk mAb,
and analyzed by anti-phosphotyrosine (Anti-Ptyr)
immunoblotting with RC20H.
To determine which domain of Crk interacts with p116, GST
fusion proteins, encoding either full-length Crk or only the Crk SH2
domain (bound to glutathione-agarose beads), were used to probe
activated T cell lysates. Anti-phosphotyrosine blotting revealed that
both GST-Crk I and GST-Crk II precipitated p116 from activated lysates
but not from non-activated lysates (Fig. 4, lanes
3-6). Moreover, GST-Crk SH2 alone precipitated equivalent
amounts of p116 compared to GST-Crk I and GST-Crk II, indicating that
the SH2 domain of Crk is responsible for its association with p116 (Fig. 4, lane 8). No p116 was precipitated from
activated lysates by either GST alone or GST-Shc SH2 fusion protein (Fig. 4, lanes 1, 2, 9, and 10).
Figure 4:
Crk SH2 domain interacts with p116 from
activated T cell lysates. GST-Crk I (lanes 3 and 4),
GST-Crk II (lanes5 and 6), GST-Crk SH2 (lanes7 and 8), and GST-Shc SH2 (lanes9 and 10) bound to glutathione-agarose beads
were added to lysates from non-activated or BYDP T cells activated by
TCR cross-linking for 1 min at 37 °C. The phosphoproteins bound to
the beads were analyzed by anti-phosphotyrosine (Anti-Ptyr)
immunoblotting.
We and others have observed that the
tyrosine-phosphorylated p116 detected in activated T cells may be
related to the p130 observed in v-Src-transformed fibroblasts (data not
shown and (28) and (29) ). Peptide maps of p116 and
p130 revealed a similar pattern indicating that p116 in T cells may be
an isoform of the p130 found in fibroblasts (data not shown and (28) and (29) ). p130 from fibroblasts has been
recently cloned (18) and was found to contain nine
tyrosine-containing motifs that represent the predicted optimal Crk SH2
binding sequence (YDXP, where X is any amino
acid)(13) . To determine whether the p116 co-precipitated with
Crk from activated T cells might represent the T cell isoform of
p130
Figure 5:
Crk-associated p116 may be related to
p130
It
has been reported that Crk, via its SH3 domain, is constitutively
associated with a novel Ras GTP/GDP exchange factor, C3G in PC12
pheochromocytoma cells(17) . To determine whether Crk can
interact with C3G in T cells, two approaches were taken. First, GST-Crk
fusion proteins were incubated with activated and non-activated T cell
lysates and immunoblotted with anti-C3G antibody. C3G was precipitated
by full-length GST-Crk I and GST-Crk II from both activated and
non-activated lysates (Fig. 6, lanes2, 3, 6, and 7). Irrespective of the activation
status, GST-Crk SH2 did not associate with C3G (Fig. 6, lanes4 and 8). Second, Crk
immunoprecipitates were immunoblotted for C3G (Fig. 7). C3G was
detected when Crk-L immunoprecipitates were immunoblotted for C3G.
However, only small amounts of C3G were found in Crk II
immunoprecipitates and none were found in Crk I immunoprecipitates (Fig. 7). These data would suggest that there is an association
of Crk-L with C3G in vivo. Because Crk I antibody interfered
with Crk-L binding to C3G, we cannot rule out an interaction of Crk I
and Crk II with C3G (data not shown).
Figure 6:
C3G associates with GST-Crk fusion
proteins. BYDP T cells were activated by TCR and CD4 cross-linking for
1 min at 37 °C and lysed with 1% Brij-96 lysis buffer. GST-Crk I
and GST-Crk II fusion proteins were incubated with non-activated and
activated BYDP lysates. Precipitates were analyzed by anti-C3G
immunoblotting. As a control, C3G was immunoprecipitated from
non-activated T cell lysates with anti-C3G antibody and immunoblotted
with the anti-C3G antibody. The arrow indicates
C3G.
Figure 7:
C3G
associates with Crk-L in vivo. BYDP T cells were activated by
TCR and CD4 cross-linking for 1 min at 37 °C and lysed with 1%
Brij-96 lysis buffer. Crk was immunoprecipated with Crk I, Crk II, or
Crk-L specific mAbs from non-activated and activated lysates.
Precipitates were analyzed by anti-C3G
immunoblotting.
Taken together, these data
suggest that, in T cells, Crk interacts with p116 via its SH2 domain
and possibly with a mediator of Ras activation, C3G via its SH3 domain.
Since phosphorylated p130
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 2893-2896
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and/or p120-Cbl. In addition, we observed that GST-Crk
fusion proteins and Crk-L bind, most likely via their SH3 domain, to
C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of
Crk with p116 and C3G strongly implicates Crk as a mediator of T cell
receptor signaling, possibly involved in Ras activation.
)domain and a SH3 domain(3, 4) , whereas
Crk II and Crk-L are composed of one SH2 domain and two SH3
domains(5) . All three Crk proteins lack an apparent catalytic
domain and belong to the recently described class of SH2/SH3-containing
adapter proteins(6, 7) , which includes
Grb2(8) , Shc(9) , Nck(10) , and the p85
subunit of phosphatidylinositol 3-kinase(11) . SH2 domains bind
to specific phosphotyrosine-containing sequences, while SH3 domains
bind to proline-rich sequences(12, 13) .)(15) , respectively. It has been
demonstrated that Crk, via its SH2 domain, interacts with both
phosphorylated paxillin and p130in
vivo(15, 16) . In addition, Crk, via its SH3
domain, interacts with two Ras guanine nucleotide exchange factors, C3G (17, 18) and mSOS(17, 19) . The
ability of Crk to simultaneously interact with the
transformation-related proteins (paxillin and
p130) via its SH2 domain and with guanine
nucleotide exchange factors via its SH3 domains suggests that Crk may
play a role in regulating the status of Ras activation. This
postulation is supported by the finding that expression of Crk proteins
carrying a point mutation in their SH3 domain (which do not interact
with C3G and mSOS) resulted in diminished Ras activation following
nerve growth factor stimulation of PC12 pheochromocytoma
cells(17) . Furthermore, overexpression of Crk in PC12 cells
leads to more rapid cellular differentiation upon nerve growth factor
or basic fibroblast growth factor stimulation(20) .
Cells
BYDP, a L3T4
Lyt2/3 murine T cell hybridoma expressing
comparable amount of retrovirus transduced human CD4 and human CD8
genes(21) , was grown in RPMI 1640 complete medium (RPMI 1640
supplemented with 5% fetal bovine serum, 5% iron-supplemented bovine
calf serum, 2 mML-glutamine, 100 units of
penicillin/streptomycin, and 2 
10
M 
-mercaptoethanol). Eighteen h prior to activation, BYDP was
placed in serum-free medium (same as complete RPMI 1640 medium, except
that 10% serum was replaced by 0.5% bovine serum albumin). Human
peripheral mononuclear cells were isolated from the whole blood by
Ficoll separation, followed by monocyte/macrophage depletion via
plastic adherence. The remaining cells were enriched for T cells by
passage through a nylon wool column. Using the anti-CD3 antibody
(OKT3), a sample of enriched T cells were analyzed by flow cytometry to
ensure that they comprised at least 80% T cells. Human peripheral T
cells were also cultured in serum-free medium for 18 h before
activation. The v-Src-transformed fibroblast cell line IV5 was a gift
from Dr. S. J. Parsons(22) .T Cell Activation
BYDP (1.5 10
cells/sample) were washed once and incubated with 1 µg of
anti-murine TCR (F23.1) and/or anti-human CD4 (OKT4D) or anti-human CD8
(OKT8) as described previously(21) . After 10 min on ice with
the stimulating antibodies, 7.5 µg of rabbit anti-mouse antibody
was added for cross-linking, and incubated for an additional 10 min on
ice. The cells were then warmed to 37 °C for the indicated times.
For human peripheral blood T cell stimulation, 3 10 cells/sample were stimulated with anti-CD3 antibody (OKT3) as
described above.Immunoprecipitations and Immunoblotting
Cells were
lysed in a buffer containing either 1% Nonidet P-40 or 1% Brij-96 and
50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM Na
VO
, 10 mM NaF, 10 mM sodium pyrophosphate, 10 µg/ml each aprotinin and leupeptin,
and 2 mM phenylmethylsulfonyl fluoride. The lysates were
precleared with protein A-Sepharose; subsequently, proteins were
immunoprecipitated with either 2 µg of anti-Crk antibody or an
equal amount of normal rabbit Ig by incubation at 4 °C for 2 h. The
beads were washed extensively with 0.1% Nonidet P-40 or 0.1% Brij-96 in
immunoprecipitation wash buffer (150 mM NaCl, 20 mM HEPES, pH 7.4, 10% glycerol, 1 mM NaVO, 5 mM NaF, 10 µg/ml each
of aprotinin and leupeptin), and the bead-bound proteins were separated
by SDS-polyacrylamide gel electrophoresis. The proteins were
transferred to a nitrocellulose membrane, immunoblotted with the
indicated antibodies, and developed by the enhanced chemiluminescence
(ECL) system (Amersham Corp.). As for precipitation with GST-Crk or
GST-Crk SH2 fusion proteins, 2-5 µg of each fusion protein
was incubated with T cell lysates. The precipitation and wash
conditions were identical to those described above.Reagents
All GST-Crk constructs were a gift from
Dr. B. J. Mayer. The GST-Shc SH2 fusion protein was a gift from Dr. T.
Pawson. The anti-Crk monoclonal antibody (mAb) and horseradish
peroxidase-coupled anti-phosphotyrosine antibody (RC20H) were purchased
from Transduction Laboratories (Lexington, KY). The anti-Crk II and
anti-Crk-L polyclonal antibodies were purchased from Santa Cruz
Biotechnologies (Santa Cruz, CA). The 4F4 anti-p130 Src substrate
(p130
) mAb was a gift from Dr. J. T.
Parsons(23) , and the anti-C3G anti-serum was a gift from Dr.
M. Matsuda(18) . The anti-phosphotyrosine mAb (4G10) was
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The
anti-c-Cbl antibody was purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA).
(Lck), Lck may be
involved in p116 phosphorylation. It has been shown that phosphorylated
p116 also interacts with the SH2 domain of another Src family tyrosine
kinase, p59
(Fyn)(28, 29) , which, in
turn, associates with the TCR 
/
and CD3 
and 
chains(30, 31) . Since p116 has been reported to
interact with the Fyn SH2 domain (30, 31) and with the
SH3 domains of both Fyn and Lck(32) , both of these kinases may
play a role, either directly or indirectly, in p116 phosphorylation.
, Crk immunoprecipitates were immunoblotted with 4F4,
a mAb that recognizes both p130 and p116. The 4F4 mAb
immunoprecipitated tyrosine-phosphorylated p116 from activated T cell
lysates and phosphorylated p130 from Src-transformed
fibroblasts (Fig. 5, left panel). p116 was detected by
4F4 immunoblotting in Crk immunoprecipitates from activated T cell
lysates (Fig. 5, right panel) but not in control Ig or
Nck immunoprecipitates (data not shown). These data suggest that p116
may also contain YDXP motifs similar to p130 and
that tyrosine phosphorylation of this motif following T cell activation
leads to its interaction with the Crk SH2 domain. It should be noted,
however, that it has been recently shown that a significant portion of
the tyrosine-phosphorylated proteins in the 116-kDa region of T cell
lysates can be immunoprecipitated by an antibody to c-Cbl(33) .
Cbl structure contains a YDXP Crk binding motif(34) ,
and it may be a component of the p116 complex that binds to Crk.
. Leftpanel, lysates from
BYDP T cells activated by TCR cross-linking for 1 min at 37 °C or
Src-transformed fibroblasts (IV5) were immunoprecipitated with
anti-p130 mAb (4F4) and immunoblotted with
anti-phosphotyrosine (Anti-Ptyr) antibody. Right
panel, Crk was immunoprecipitated with the Crk mAb from BYDP cells
activated for 1 min by TCR cross-linking. Precipitates were
immunoblotted with 4F4.
in fibroblasts has been
localized to the plasma membrane (15) and T cell p116 has been
shown to interact with TCR-associated Fyn(28, 29) ,
the Crk-p116 inter-action may serve to shuttle C3G to the membrane.
This, in turn, may contribute to TCR-mediated, tyrosine
kinase-dependent Ras activation. In T cells, three other proteins: Shc
and p36 (via their interaction with Grb2 and
mSOS)(27, 35) , and Vav (through its intrinsic
nucleotide exchange activity)(36) , are also implicated in Ras
activation. Since the Crk SH3 domain can also interact with
mSOS(17, 19) , Crk might also be able to activate the
Ras pathway via mSOS. Whether these are distinct pathways of Ras
activation or whether they represent pathways employed under different
stimulation conditions remains to be determined. While the precise role
of Crk in TCR-mediated signaling remains to be established, our data
strongly suggest an important role for Crk in T cell activation.
)
We are very grateful to Dr. M. Matsuda for the
anti-C3G antibody, Dr. B. J. Mayer for the GST-Crk constructs,
Dr. T. Pawson for GST-Shc SH2 fusion protein, Dr. S. J. Parsons for the
IV5 Src-transformed fibroblast cell line, and to Dr. J. T. Parsons for
the 4F4 mAb. We thank L. Samelson and H. Meissner for helpful
discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y.-C. Liu, Y. Liu, C. Elly, H. Yoshida, S. Lipkowitz, and A. Altman Serine Phosphorylation of Cbl Induced by Phorbol Ester Enhances Its Association with 14-3-3Proteins in T Cells via a Novel Serine-rich 14-3-3-binding Motif J. Biol. Chem., April 11, 1997; 272(15): 9979 - 9985. [Abstract] [Full Text] [PDF]
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C. J. Fitzer-Attas, D. G. Schindler, T. Waks, and Z. Eshhar Direct T Cell Activation by Chimeric Single Chain Fv-Syk Promotes Syk-Cbl Association and Cbl Phosphorylation J. Biol. Chem., March 28, 1997; 272(13): 8551 - 8557. [Abstract] [Full Text] [PDF]
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Y.-C. Liu, C. Elly, W. Y. Langdon, and A. Altman Ras-dependent, Ca2+-stimulated Activation of Nuclear Factor of Activated T Cells by a Constitutively Active Cbl Mutant in T Cells J. Biol. Chem., January 3, 1997; 272(1): 168 - 173. [Abstract] [Full Text] [PDF]
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A. Y. Tsygankov, S. Mahajan, J. E. Fincke, and J. B. Bolen Specific Association of Tyrosine-phosphorylated c-Cbl with Fyn Tyrosine Kinase in T Cells J. Biol. Chem., October 25, 1996; 271(43): 27130 - 27137. [Abstract] [Full Text] [PDF]
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JeraldA. Donovan, Y. Ota, WallaceY. Langdon, and LawrenceE. Samelson Regulation of the Association of p120cbl with Grb2 in Jurkat T Cells J. Biol. Chem., October 18, 1996; 271(42): 26369 - 26374. [Abstract] [Full Text] [PDF]
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K. K. Lee-Fruman, T. L. Collins, and S. J. Burakoff Role of the Lck Src Homology 2and 3Domains in Protein Tyrosine Phosphorylation J. Biol. Chem., October 4, 1996; 271(40): 25003 - 25010. [Abstract] [Full Text] [PDF]
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K. A. Reedquist, T. Fukazawa, G. Panchamoorthy, W. Y. Langdon, S. E. Shoelson, B. J. Druker, and H. Band Stimulation through the T Cell Receptor Induces Cbl Association with Crk Proteins and the Guanine Nucleotide Exchange Protein C3G J. Biol. Chem., April 5, 1996; 271(14): 8435 - 8442. [Abstract] [Full Text] [PDF]
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Lás. Buday, A. Khwaja, S. Sipeki, A. Faragó, and J. Downward Interactions of Cbl with Two Adaptor Proteins, Grb2 and Crk, upon T Cell Activation J. Biol. Chem., March 15, 1996; 271(11): 6159 - 6163. [Abstract] [Full Text] [PDF]
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