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INTRODUCTION |
Engagement of the T cell antigen receptor
(TCR)1 triggers signal
transduction pathways that directly regulate T cell activation and
differentiation (1, 2). Although many of the individual components of
the TCR-linked signaling pathway are physically separated in resting
cells, upon engagement of the receptor, they reassemble into functional
complexes at the site of contact with major histocompatibility
complex-presenting peptide antigen on the surface of the
antigen-presenting cells (3). The reassembly process directs enzymes,
their corresponding substrates, and additional effector molecules to
the receptor site, in a temporally and spatially regulated manner,
which ensures an efficient signaling leading to cell activation. The
same mechanism ensures that the level of signaling in unstimulated
cells remains below the critical threshold required for cell activation.
The earliest detectable biochemical event in activated T cells is the
phosphorylation of multiple protein substrates on tyrosine residues.
Therefore, protein tyrosine kinases (PTKs) that mediate this activity
play a critical role in the early phases of the activation response by
up-regulating critical enzymes and promoting the assembly of the
multimolecular complexes. Non-receptor PTKs that mediate these
functions in T cells include the Src family members, Lck and Fyn, and
the Syk family members, ZAP-70 and Syk (1, 2). A fraction of Fyn and
Lck, which are constitutively associated with the TCR and CD4/CD8
cytoplasmic tails, respectively, appears to phosphorylate critical
tyrosine residues within the immunoreceptor tyrosine-based activation
motifs (ITAMs) on distinct TCR subunits (4). ZAP-70 and Syk then
recruit to the phosphorylated ITAMs and interact with two
phosphotyrosyl residues within an ITAM via their tandem SH2 domains.
Only then can ZAP-70 undergo tyrosine phosphorylation and become
enzymatically active (5, 6). The subsequent tyrosine phosphorylation of
additional cellular substrates, including LAT, phospholipase C
1,
Vav, SLP-76, Shc, Cbl, and Pyk2, function to couple the receptor to its
signaling pathways and regulate the activation response.
Members of the group of adapter proteins, which include Crk, Grb2, Nck,
Grap, and Shc, consist primarily of SH2 and SH3 protein-protein interaction domains. They are involved in the control of various cellular processes linked to cell growth and differentiation. Crk
adapter proteins have been implicated in signaling pathways leading to
cell growth (7), migration (8), differentiation (9), apoptosis (10),
and transformation (11). Although the physiological role of Crk
proteins is largely unknown, studies have shown their involvement in
signaling via diverse membrane receptors, including those of integrins
(12), interleukins (13), and growth factors (13-15). Furthermore, Crk
proteins play a role in signaling via antigen receptors in B (16-18)
and T (19-22) lymphocytes. Recent data suggest that the involvement of
Crk proteins in various signaling pathways is mediated by their ability
to up-regulate the activity of small GTP-binding proteins, such as Ras
(15, 23, 24), Rap1 (25, 26), and Rho (27), and activate a selective
mitogen-activated protein kinase cascade which is controlled by c-Jun
NH2-terminal kinase (23, 28).
Crk proteins have also been reported to interact with a variety of
cellular proteins. These include tyrosine-phosphorylated proteins,
such as the multidomain docking proteins, Cas (Crk-associated substrate, p130; see Ref. 29) and HEF1 (human enhancer for
filamentation 1; see Ref. 30), tyrosine kinase receptors such as the
PDGF-R (31), HEK2 (32), and Ret (33), and additional proteins including paxillin (p70; see Ref. 34), Cbl (p120; see Ref. 19), and IRS-1
(insulin receptor substrate-1; see Ref. 35), most of which interact
with the Crk SH2 domain. Furthermore, Crk can interact via its SH3
domain with proline-rich sequences in the guanine nucleotide exchange
factors, C3G (36) and Sos (24), in Abl (37) and Arg PTKs (38), in
addition to proteins that include DOCK180 (39) and EPS15 (40).
Nevertheless, the precise role of Crk in the relevant signaling
pathways has not been defined.
To analyze further the involvement of Crk proteins in the early
activation events in T lymphocytes, we searched for T cell-derived tyrosyl phosphoproteins that associate with Crk in an
activation-dependent manner. In this report, we describe
experiments showing that the ZAP-70 PTK interacts with Crk in activated
T lymphocytes. We also provide data to establish the mechanism of
interaction between the two molecules and determine the activity and
subcellular location of the Crk-associated ZAP-70.
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EXPERIMENTAL PROCEDURES |
Reagents--
Phytohemagglutinin, histopaque-1077,
glutathione S-transferase (GST), phenylmethylsulfonyl
fluoride, aprotinin, and leupeptin were from Sigma. AEBSF was from ICN
Biomedicals, Inc. (Aurora, OH). Human recombinant IL-2 was a gift from
Hoffmann-LaRoche. Nitrocellulose membranes were from Schleicher & Schuell; ECL was from Amersham Pharmacia Biotech (Buckinghamshire, UK);
protein A-Sepharose was from Amersham Pharmacia Biotech (Uppsala,
Sweden); and [-32P]ATP (3000 Ci/mmol) was from Rotem
Industries, Ltd. (Beer Sheva, Israel). The cdb3/T7-7 expression vector,
containing the cytoplasmic domain (residues 1-379) of human
erythrocyte band 3 (CFB3) gene, was a gift of Dr. P. S. Low (Purdue University, IN). This construct was used to transform
Escherichia coli BL21 (DE3)(pLysS) (Novagen, Madison, WI),
and expression and purification of CFB3 were performed essentially as
described (41).
Antibodies--
Anti-phosphotyrosine (PY) mAbs were either from
culture supernatants of PY.72.10.5 hybridoma (a gift from Dr. B. Sefton, the Salk Institute, La Jolla, CA) or 4G10 mAb from Upstate
Biotechnology Inc. (Lake Placid, NY). Cross-reactive anti-Crk-I/Crk-II
polyclonal antiserum was raised in rabbits by immunization with a
GST-Crk-II fusion protein, and a mouse mAb specific to Crk-I/Crk-II was
from Transduction Laboratories (Lexington, KY). Rabbit anti-ZAP-70 polyclonal antiserum was raised against GST fusion protein containing amino acids 255-345 of human ZAP-70, a gift of Dr. J. B. Bolen (Bristol-Myers Squibb Co.) (41). Affinity purified anti-human CD3 mAb
was obtained from ascites of the OKT3 hybridoma (obtained from the
ATCC, Rockville, MD), and the TCR
-chain-specific mAb C305 was a gift
of Dr. A. Weiss (University of California, San Francisco, CA). A mouse
mAb anti-GST was from Santa Cruz Biotechnology, Inc. Horseradish
peroxidase (HRP)-conjugated sheep anti-mouse or donkey anti-rabbit,
immunoglobulin Abs, and HRP-conjugated protein A were from Amersham
Pharmacia Biotech.
GST Fusion Proteins--
A plasmid containing the GST-CrkL was a
gift of Dr. B. Druker (Oregon Health Sciences Center, Portland, OR),
and plasmids containing GST fused to Crk-I, Crk-II, or individual
Crk-II domains were gifts of Dr. M. Matsuda (National Institute of
Health, Tokyo, Japan). pGEX plasmids were used to transform E. coli DH5
cells (Life Technologies, Inc.). After induction of
protein expression with 0.1 mM
isopropyl-1-thio--D-galactopyranoside (Promega, Madison, WI) for 2-4 h, the bacteria were resuspended in a lysis buffer containing 50 mM Tris/HCl, pH 8.0, 100 mM NaCl,
and 1 mM phenylmethylsulfonyl fluoride and further
disrupted by sonication. Following centrifugation at 10,000 × g for 20 min, the induced proteins were adsorbed to immobilized glutathione-agarose. Soluble GST fusion proteins were obtained by elution with 5 mM reduced glutathione (Roche
Molecular Biochemicals, Mannheim, Germany) in 50 mM
Tris/HCl, pH 8.0.
For in vitro binding assays, bead-adsorbed GST or GST fusion
proteins (5 µg/sample) were incubated with cell lysates at 4 °C on
a rotator for 1 h. The beads were then washed 3 times in lysis
buffer, and bound proteins were either eluted and subjected to SDS-PAGE
under reducing conditions followed by immunoblotting or tested in an
in vitro kinase assay.
Cell Culture and Stimulation--
Human leukemic Jurkat T
cells, Jurkat-TAg cells which stably express the simian virus
40-derived large T antigen, and Jurkat-derived mutant cell lines, JCaM1
and J4501, that are defective in expression of Lck or CD45,
respectively, were maintained at a logarithmic growth phase in complete
RPMI (RPMI 1640 supplemented with 5% heat-inactivated fetal calf
serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin (all from Biological Industries, Beit Haemek, Israel), and 5 × 10
5 M
-mercaptoethanol (Sigma)) in 75-cm2 growth-area tissue
culture flasks (Cell-Cult, Sterilin Limited, Feltham, UK) in an
atmosphere of 7.5% CO2, at 37 °C. Peripheral blood
lymphocytes (PBL) were obtained by histopaque gradient centrifugation of heparinized blood from healthy volunteers. Enriched population of
preactivated and rested PBL T cells were obtained by cell culture (1 × 106/ml) in 10% fetal calf serum-containing
complete RPMI in the presence of 5 µg/ml phytohemagglutinin in
75-cm2 growth-area tissue culture flasks (50 ml/flask).
Human recombinant IL-2 (20 units/ml) was added after 72 h of
culture, and cells were maintained in culture for 6 more days by the
addition of IL-2 (20 units/ml) once every 2 days.
Jurkat or PBL T cells (10 × 106/100 µl) were
stimulated with freshly prepared 1% pervanadate (10 mM
Na3VO4 containing 1%
H2O2) for 30 min at 37 °C. Ab-mediated
cross-linking of the TCR/CD3 was performed by incubating Jurkat or PBL
T cells (10 × 106/100 µl) with C305 mAb, or OKT3
mAb, respectively, for 10 min on ice. A secondary cross-linking rabbit
anti-mouse Ig Ab was then added for 10 min on ice, followed by transfer
to 37 °C and incubation for 2 min.
Mammalian Expression Vectors and Transient Transfection of
Cells--
The CRK-II cDNA in
pcDL-SR296(BglII) mammalian expression vector
(pVCrk-II) was a gift of Dr. Matsuda (42). For transfection, the Jurkat-TAg cells were washed three times in supplement-free RPMI
1640, resuspended at 5 × 107 cells per ml in
unsupplemented medium, and aliquoted into 0.4-cm-gap Gene Pulser
cuvettes (Bio-Rad) (2 × 107 cells/400 µl/cuvette).
Plasmid DNA (10 µg/group) was added, and the cells were
electroporated using a Bio-Rad Gene Pulser (250 volts, 950 microfarads). The cells were then cultured in 13 ml of complete RPMI
1640 in T25 tissue culture flasks for 48 h.
Preparation of Cell Lysates and Immunoprecipitation--
Cell
lysates were prepared by resuspension of cells in a lysis buffer
containing 25 mM Tris/HCl, pH 7.5, 150 mM NaCl,
5 mM EDTA, 1 mM Na3VO4,
50 mM NaF, 10 µg/ml each of leupeptin and aprotinin, 2 mM AEBSF, and 1% Triton X-100, followed by a 20-min
incubation on ice. Lysates were centrifuged at 13,000 × g for 30 min at 4 °C, and the nuclear free supernatants
were mixed with equal volumes of 2× SDS sample buffer, vortexed,
incubated at 100 °C for 5 min, and analyzed by SDS-PAGE. Cytosol and
particulate fractions were prepared by resuspending the cells in buffer
A (20 mM Tris/HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 10 mM -mercaptoethanol, 10 µg/ml each of leupeptin and aprotinin, and 2 mM AEBSF) and
repeatedly aspirating them through a 1-ml syringe with a 26-gauge
needle for 20 s. Cell lysates were centrifuged at 400 × g for 5 min; nuclear pellets were removed, and lysates were
recentrifuged at 13,000 × g. Supernatants (cytosolic
fractions) were transferred to a second set of microcentrifuge tubes,
Triton X-100 was added up to a 1% final concentration, and samples
were either mixed with 5× SDS sample buffer (4:1, v/v), or used for
immunoprecipitation. Pellets were washed once in buffer A, resuspended
in buffer A plus 1% Triton X-100 (in the original volume used for the
lysis), incubated for 30 min on ice, and centrifuged at 13,000 × g for 20 min. Supernatants (particulate fractions) were
either mixed with 5× SDS sample buffer (4:1, v/v) or used for immunoprecipitation.
Immunoprecipitation was performed by using an optimal dilution of
polyclonal antisera or mAbs that were preabsorbed on protein A-Sepharose beads for 2 h at 4 °C. Excess Abs were removed by 3 washes in cold phosphate-buffered saline, and Ab-coated beads were
incubated with cell lysates for 2-3 h at 4 °C. Immune complexes were precipitated by centrifugation followed by extensive washing in a
lysis buffer. Immunoprecipitated proteins were then either fractionated
by SDS-PAGE and immunoblotted or subjected to an in vitro
kinase assay.
Electrophoresis and Immunoblotting--
Samples of cell lysates,
GST fusion protein, GST fusion protein-bound molecules, or Ab
immunoprecipitates were resolved by electrophoresis on 10% acrylamide
gels using Bio-Rad Mini-PROTEAN II Cell. Proteins in the gels were
either stained with Coomassie Brilliant Blue (Sigma) or blotted onto
nitrocellulose membranes (Schleicher & Schuell) at 100 V for 45 min in
a Bio-Rad Mini Trans-Blot transfer cell. After 1 h blocking at
37 °C with 3% bovine serum albumin in phosphate-buffered saline,
nitrocellulose membranes were incubated with the indicated primary Abs,
followed by incubation with HRP-conjugated sheep anti-mouse, or donkey
anti-rabbit, Ig, or with HRP-conjugated protein A (Amersham Pharmacia
Biotech). Immunoreactive proteins were visualized using an ECL reagent
(Amersham Pharmacia Biotech) and autoradiography.
Far Western Analysis--
To determine direct interaction of
either Crk-II or selected Crk-II domains with the electrophoresed,
nitrocellulose-bound proteins, the SDS-PAGE and blotting were performed
as described above, followed by blocking of the membranes with
phosphate-buffered saline containing 3% bovine serum albumin and 0.1%
Tween 20. Membranes were then incubated overnight at 4 °C with a
blocking buffer containing 10 µg/ml of the indicated GST fusion
protein, or GST, as a negative control. Bound GST proteins were
detected by incubation of the membrane with a mouse anti-GST mAb for
1 h, followed by an HRP-conjugated sheep anti-mouse Ig and ECL development.
ZAP-70 Kinase Assay--
ZAP-70 immunoprecipitates or cell
lysate proteins adsorbed to immobilized GST fusion protein were washed
twice in a lysis buffer followed by an additional wash in a kinase
reaction buffer (25 mM HEPES, pH 7.3, 0.1% Nonidet P-40,
10 mM MnCl2, 1 mM
Na3VO4, 50 mM NaF). They were then
resuspended in a reaction buffer, with or without 1 µg of CFB3.
Kinase reaction, in a total volume of 15 µl, was initiated by the
addition of 5 µCi of 10 µM [-32P]ATP
(at 3000 Ci/mmol) and incubated for 10 min at 30 °C. Reaction was
terminated by the addition of 5× SDS sample buffer (4:1, v/v); samples
were vortexed and boiled for 5 min, and phosphoproteins were resolved
by SDS-PAGE on 10% acrylamide gels. Phosphoproteins were blotted onto
nitrocellulose membranes and visualized by autoradiography by exposure
to Kodak XAR-5 x-ray film at 
70 °C with an intensifying screen.
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RESULTS |
Crk Adapter Proteins Associate with ZAP-70 from Activated Jurkat T
Cells--
Adapter proteins that consist of SH2 and SH3 domains play
critical roles in the assembly of multimolecular signaling complexes during the early phases of cell activation response. We analyzed the
involvement of Crk adapter proteins in the regulation of T cell
activation, and because many of the protein-protein interaction events
are regulated by tyrosine phosphorylation of signaling molecules, we
questioned whether Crk proteins interact with tyrosine-phosphorylated proteins in activated T cells. Lysates of non-activated or
pervanadate-stimulated human Jurkat T cells were incubated with
bead-immobilized GST-Crk fusion proteins. After separation of the bound
proteins by SDS-PAGE, the proteins were transblotted to nitrocellulose
membranes, and tyrosyl phosphoproteins were identified using
phosphotyrosine-specific mAbs. All three Crk adapter proteins were
found to pull down numerous protein bands that reacted with
anti-phosphotyrosine (Fig.
1A). One of the observed
protein bands, at the range of 70 kDa, corresponded to the molecular
mass of ZAP-70 PTK, which is a key enzyme in T cell activation.
Stripping and reblotting of the membrane with anti-ZAP-70 Abs indicated
that Crk interacted with ZAP-70 (Fig. 1A, 6th, 8th, and
10th lanes) and that the association occurred in lysates of
activated but not of non-activated T cells (Fig. 1B,
6th, 8th, and 10th lanes, versus
5th, 7th, and 9th lanes, respectively). To
examine whether this protein-protein interaction pattern is selective
for Crk, we compared it with the pattern of tyrosyl phosphoproteins
that associates with two other adapter proteins, Grb2 and Nck, which
are also expressed in T cells. Although the overall pattern of tyrosyl
phosphoproteins that interact with Crk and Grb2 was not drastically
different, ZAP-70 was found to associate with Crk adapter proteins but
not with Grb2 or Nck (Fig. 1B, 5-10th versus
11-14th lanes).
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Fig. 1.
. Fusion proteins of GST-Crk, but
not GST-Grb2 or -Nck, associate in vitro with
tyrosine-phosphorylated ZAP-70 from a lysate of pervanadate-stimulated
Jurkat T cells. Jurkat cells (4 × 107/group)
were incubated with 1% pervanadate
(perVO4) for 30 min at
37 °C followed by lysis and incubation of the lysate with 5 µg of
GST or GST fusion proteins immobilized to glutathione-agarose beads.
After 1 h of incubation on a rotator at 4 °C, the beads were
washed, and bound proteins were eluted and subjected to SDS-PAGE under
reducing conditions. Proteins were then electroblotted (IB)
onto nitrocellulose membranes, and tyrosine-phosphorylated proteins
were visualized by reaction with anti-phosphotyrosine (pY)
mAbs and development with immunoperoxidase ECL detection system and
autoradiography (A). After stripping of the nitrocellulose
membranes from bound Abs, membranes were reblotted with Abs specific
for ZAP-70 (B). Molecular size markers (in kilodalton) are
indicated on the left. The position of the anti-ZAP-70
reactive 70-kDa protein band is indicated by an arrowhead.
Results are representative of five experiments.
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Depletion of ZAP-70 from the lysate of activated T cells by repeated
absorption with bead-immobilized anti-ZAP-70 Abs confirmed that the
Crk-associated 70-kDa protein is ZAP-70 and is not a distinct 70-kDa
protein that reacts with the secondary anti-rabbit Ab or directly
interacts with the ECL (Fig. 2).
Furthermore, the GST-Crk-II fusion protein pulled down a
tyrosine-phosphorylated 70-kDa protein only from wild-type Jurkat cells
but not from its ZAP-70-deficient mutant, P116 (not shown).
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Fig. 2.
The 70-kDa phosphoprotein from activated
Jurkat T cells which interacts with GST-Crk is depleted by pretreatment
with anti-ZAP-70 Abs. A, Jurkat cells (4 × 107/group) were either not treated or were incubated with
1% pervanadate (perVO4) for 30 min at 37 °C
followed by treatment with a lysis buffer. Cell lysate supernatants
were either not treated or underwent three consecutive
immunoprecipitation steps with protein A-agarose-bound anti-ZAP-70 Abs.
Supernatants were then incubated with protein A-agarose-bound
anti-ZAP-70 Abs (1st lane), 5 µg of GST
(2nd lane), or 5 µg of GST-Crk-II immobilized
to glutathione-agarose beads (3rd and 4th lanes).
After 1 h of incubation on a rotator at 4 °C, the beads were
washed, and bound proteins were eluted and subjected to SDS-PAGE and
immunoblotting (IB) with anti-ZAP-70 Abs, as described in
the legend to Fig. 1. B, supernatants from activated Jurkat
T cell lysates that were subjected to 1, 2, or 3 sequential
immunoprecipitations with anti-ZAP-70 Abs were analyzed by SDS-PAGE
under reducing conditions, electroblotted onto nitrocellulose
membranes, and immunoblotted with anti-ZAP-70 Abs, as indicated above.
Molecular size markers (in kilodalton) are indicated on the
right, and the position of the anti-ZAP-70 reactive 70-kDa
protein band is indicated by an arrowhead. Similar results
were obtained in two other experiments.
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The human ZAP-70 protein possesses 31 tyrosine residues that are
potential phosphorylation sites for PTKs in pervanadate-activated T
cells. One or more of these phosphotyrosyl residues may possibly mediate binding to the Crk-SH2 but may not necessarily be a potential site in TCR-activated T cells. We therefore tested whether
cross-linking of the TCR on Jurkat T cells would also induce
phosphorylation of ZAP-70 at a site that allows interaction with Crk.
Activation of TCR on Jurkat cells was induced by incubation with a TCR
V-chain-specific mAb, C305, followed by analysis of the interaction
of cell lysate proteins with various GST-Crk fusion proteins. The
results demonstrated that C305 stimulation of Jurkat cells induced the
association of ZAP-70 with Crk and that the binding affinity to the
three Crk proteins was markedly distinct, indicating a hierarchy of Crk-II 
CrkL 
Crk-I (Fig.
3A). This was based on
observations of autoradiograms obtained after extended periods of
exposure (not shown). Many of the protein bands observed in the
anti-ZAP-70 immunoblots reflected the GST portion of the fusion
proteins and their degradation products that react with Abs against the
GST portion of the immunogen.
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Fig. 3.
. GST-Crk associates in vitro
with ZAP-70 from lysates of TCR-stimulated Jurkat T cells and
peripheral blood T cells. Jurkat cells (4 × 107/group, A) or peripheral blood lymphocytes
(4 × 107/group, B and C) were
incubated with either TCR V-chain-specific mAb, C305 (A),
or anti-CD3, OKT3 mAb (B and C), for 2 min at
37 °C, followed by lysis and incubation of the lysate with 5 µg of
GST or GST fusion proteins immobilized to glutathione-agarose beads.
After 1 h of incubation on a rotator at 4 °C, the beads were
washed, and bound proteins were eluted and subjected to SDS-PAGE under
reducing conditions. Proteins were then electroblotted onto
nitrocellulose membranes followed by incubation with anti-ZAP-70 Abs
and immunoperoxidase ECL detection system. Immunoreactive proteins were
visualized by autoradiography (A and B). After
stripping, the nitrocellulose membranes were reblotted with
phosphotyrosine-specific mAbs (C). Molecular size markers
(in kilodalton) are indicated on the left. The position of
the anti-ZAP-70 reactive 70-kDa protein band is marked with an
arrowhead. Results are representative of four experiments
performed on Jurkat cells (A) and three experiments
performed on peripheral blood lymphocytes obtained from three
independent donors (B). pY,
phosphotyrosine.
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To analyze further whether ZAP-70-Crk association represents a general
phenomenon in activated T cells, or perhaps an event unique for Jurkat
cells or leukemic cells, we performed a similar analysis on lymphocytes
from human peripheral blood. Because ZAP-70 is expressed in T, but not
B, lymphocytes, we used unseparated peripheral blood lymphocytes
stimulated with polyclonal anti-CD3 (OKT3) mAbs. As in Jurkat cells,
ZAP-70 from OKT3-stimulated peripheral blood T lymphocytes interacted
with GST-Crk (Fig. 3B), and distinct Crk proteins exhibited
a hierarchy of binding affinities identical to the one observed in
Jurkat cells (Fig. 3A). Stripping and re-blotting of the
membrane with Tyr(P)-specific mAbs confirmed that a tyrosyl phosphoprotein band with a molecular mass of 70 kDa was pulled down by
GST-Crk from a lysate of OKT3-stimulated, but not resting, PBL (Fig.
3C).
To demonstrate the interaction between ZAP-70 and Crk in activated T
cells in a more direct way, Jurkat cell lysates were subjected to
immunoprecipitation with either anti-Crk or anti-ZAP-70 Abs and
blotting with the reciprocal combination of Abs. We found that Crk mAbs
co-immunoprecipitated ZAP-70 from a lysate of activated but not
non-activated Jurkat cells (not shown). Because of the relatively low
stoichiometry of binding obtained in this assay, we repeated the
immunoprecipitation in Jurkat-TAg cells that transiently overexpressed
the Crk-II cDNA. As previously, anti-Crk mAbs were found to
co-immunoprecipitate ZAP-70; association between the two proteins was
specific for activated, but not non-activated, T cells (Fig.
4). The reciprocal immunoprecipitation
with anti-ZAP-70 Abs and immunoblotting with anti-Crk did not yield
conclusive results. This could be due to the fact that the anti-ZAP-70
polyclonal antiserum cross-reacts with additional proteins that
interfere with the analysis and/or the possibility that anti-ZAP-70 Abs compete with Crk in binding to the same epitopes on the ZAP-70 molecule.
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Fig. 4.
ZAP-70 from a lysate of
pervanadate-stimulated Jurkat T cells co-immunoprecipitates
(IP) with Crk proteins. Jurkat cells (2 × 107/cuvette) were electroporated with 10 µg of
pVCrk-II DNA, cultured for an additional 48 h at
37 °C, and then stimulated with 1% pervanadate
(perVO4) for 30 min at 37 °C. Cells were
treated with lysis buffer, and lysates were incubated with 2 µg of
anti-Crk mAbs (or non-relevant serum (NRS), as a control)
plus protein A-conjugated agarose beads. After 1 h of incubation
on a rotator at 4 °C, the beads were washed, and bound proteins were
eluted and subjected to SDS-PAGE under reducing conditions. Proteins
were then electroblotted (IB) onto nitrocellulose membranes,
and ZAP-70 proteins (A) were visualized by reaction with
specific Abs and an immunoperoxidase ECL detection system followed by
autoradiography. After stripping, the nitrocellulose membranes were
reblotted with Abs specific for Crk (B). Molecular size
markers (in kilodalton) are indicated on the left, and the
position of ZAP-70 and Crk protein bands is indicated by
arrowheads. Results are representative of three
experiments.
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ZAP-70 Association with Crk Is Mediated by Direct Physical
Interaction of ZAP-70 Phosphotyrosine-containing Sequences with the
Crk-SH2 Domain--
The apparent dependence of association between
ZAP-70 and Crk on the activation stage of the T cells suggested that
this transient event is regulated by a PTK that phosphorylates ZAP-70
and increases its affinity to the Crk-SH2 domain. A PTK candidate for
this function is the Src family member, Lck, which was shown to
function as an upstream regulator of ZAP-70 (43). In order to test the
involvement of Lck in the regulation of ZAP-70 association with Crk, we
used the JCaM1 cells, a Jurkat mutant subline that is genetically
deficient in Lck. Anti-ZAP-70 blot (Fig.
5B, lower panel) demonstrated
that JCaM1 cells possess ZAP-70 protein at levels indistinguishable from those observed in wild-type Jurkat cells. Absence of Lck resulted
in a complete lack of activation-dependent tyrosine
phosphorylation of ZAP-70 (Fig. 5B, upper panel), which
correlates with the lack of association of ZAP-70 with GST-Crk-II (Fig.
5A).
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Fig. 5.
ZAP-70 interaction with Crk-II is dependent
on Lck and requires phosphorylation of tyrosine residues.
Wild-type Jurkat cells and two Jurkat-derived mutant sublines, JCaM1
and J4501, were either not stimulated or incubated with 1% pervanadate
(perVO4) for 30 min at 37 °C, followed by
lysis, and incubation of the lysate (4 × 107 cell eq
per group) with 5 µg of GST or GST-Crk-II fusion proteins immobilized
to glutathione-agarose beads. After 1 h of incubation on a rotator
at 4 °C, the beads were washed, and bound proteins were eluted and
subjected to SDS-PAGE under reducing conditions. Proteins were then
electroblotted onto nitrocellulose membranes, and ZAP-70 was visualized
by reaction with specific Abs, development with immunoperoxidase ECL
detection system, and autoradiography (A). To quantitate
ZAP-70 in total cell lysates and determine its tyrosine phosphorylation
state, ZAP-70 was immunoprecipitated from a second set of aliquots of
the cell lysates (4 × 107 cell eq per group) and
immunoblotted (IB) with anti-ZAP-70 Abs (B, lower
panel), followed by stripping of the membranes and reblotting with
anti-Tyr(P) (anti-pY) mAbs (B, upper panel).
Tyrosine phosphorylation levels of proteins in total cell lysates were
determined on a third aliquot of cell lysates (0.5 × 106 cell eq per group) by immunoblotting with anti-Tyr(P)
mAbs (C). Molecular size markers (in kilodalton) are
indicated on the left. The position of ZAP-70 is indicated
by an arrowhead. Results are representative of three
separate experiments using the three different cell lines.
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A second mutant Jurkat subline, termed J4501, that was tested lacks the
CD45 protein tyrosine phosphatase and is impaired in TCR-linked signal
transduction because of the critical role of CD45 in dephosphorylating
essential effector molecules during the early phase of the activation
response (44). Even though tyrosine phosphorylation levels of multiple
protein bands were reduced or even completely absent in activated J4501
cells (Fig. 5C), the levels of tyrosine-phosphorylated
Lck and ZAP-70 in J4501 appeared to be comparable to those of wild-type
Jurkat T cells (Fig. 5C). Accordingly, pervanadate
stimulation of J4501 cells resulted in association of ZAP-70 with
GST-Crk-II (Fig. 5A). These results indicate that ZAP-70
phosphotyrosyl-containing sequences are involved in ZAP-70 interaction
with Crk-II and suggest that Lck plays a critical regulatory role in
this association. Furthermore, the requirement of ZAP-70 phosphotyrosyl
residues in the interaction suggests the involvement of the Crk-SH2
domain. This has been substantiated in binding studies showing that the
Crk-SH2 domain (Fig. 6, 5th
lane) was sufficient for mediating the interaction with ZAP-70
from a lysate of activated Jurkat T cells, whereas neither of the two
Crk-SH3 domains (Fig. 6, 6th and 7th lanes) could
bind ZAP-70.
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Fig. 6.
Interaction of Crk-II with
tyrosine-phosphorylated ZAP-70 is mediated by the Crk-II-SH2
domain. Jurkat cells (4 × 107/group) were either
left unstimulated or incubated with 1% pervanadate
(perVO4) for 30 min at 37 °C, followed by
lysis and incubation of the lysate with 5 µg of GST or GST fusion
proteins immobilized to glutathione-agarose beads. After 1 h of
incubation on a rotator at 4 °C, the beads were washed, and bound
proteins were eluted and subjected to SDS-PAGE under reducing
conditions on two parallel gels. Proteins were then electroblotted
(IB) onto nitrocellulose membranes and ZAP-70 (upper
panel), or tyrosine-phosphorylated proteins (lower
panel) were visualized by reaction with the appropriate Abs,
development with immunoperoxidase ECL detection system, and
autoradiography. Total cell lysate of non-activated and
pervanadate-stimulated cells (0.5 × 106/group) is
included as a reference (lanes 1 and 2).
Molecular size markers (in kilodalton) are indicated on the
left. The position of ZAP-70 is indicated by an
arrowhead. Results are representative of four
experiments.
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The results thus far indicated that the interaction between ZAP-70 and
Crk is dependent on cell activation and tyrosine phosphorylation of
ZAP-70 and mediated by the Crk-SH2 domain. However, it is yet unclear
whether the two proteins interact via a direct physical contact or
through a third party mediator molecule which associate simultaneously
with ZAP-70 and Crk. To distinguish between these two possibilities, we
used a far Western blot analysis (overlay assay) in which direct
binding of soluble fusion proteins to immunoblotted ZAP-70 was tested.
We found that GST fusion proteins containing either the entire Crk-II
molecule (Fig. 7B) or the
isolated Crk-SH2 domain (Fig. 7C) directly interacted with
ZAP-70 from pervanadate-stimulated Jurkat T cells. Despite the fact
that similar levels of ZAP-70 were immunoprecipitated from
non-activated or activated Jurkat cells (Fig. 7D),
GST-Crk-II and GST-Crk-II-SH2 interacted with ZAP-70 from activated but
not from non-activated T cells (Fig. 7, B and C).
Neither Crk-SH3(N) nor Crk-SH3(C) interacted with ZAP-70 in this assay
(not shown).
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Fig. 7.
Physical binding of ZAP-70 to the Crk-SH2
domain is mediated by direct interaction. Protein A-agarose-bound
anti-ZAP-70 Abs or normal rabbit serum (NRS), as a negative
control, were used for immunoprecipitation (IP) of proteins
from nuclei-free lysates of either unstimulated or pervanadate
(perVO4)-stimulated Jurkat cells (4 × 107/group). After 1 h incubation on a rotator at
4 °C, the beads were spun down, washed three times in a lysis
buffer, and subjected to SDS-PAGE under reducing conditions, followed
by electroblotting (IB) onto nitrocellulose membranes.
Membranes were then reacted with anti-phosphotyrosine (pY)
mAbs (A), soluble GST-Crk-II (B) or
GST-Crk-II-SH2 (C) fusion proteins, or anti-ZAP-70 Abs
(D). Membrane-bound GST fusion proteins were detected by
incubation with mouse anti-GST mAbs. Immunoreactive protein bands were
visualized by reaction with an HRP-conjugated secondary Ab and
development with an immunoperoxidase ECL detection system followed by
autoradiography. Anti-Tyr(P) immunoblot of total cells lysate of
non-activated and activated Jurkat cells (0.5 × 106/group) served to control the efficiency of activation
(A, right panel). Molecular size markers (in kilodalton) are
indicated on the right, and the position of the ZAP-70
protein band is indicated by an arrowhead. Results are
representative of three experiments.
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The Crk-associated ZAP-70 from Activated Jurkat T Cells Is
Enzymatically Active--
The analysis of ZAP-70-deficient human SCID
patients has established that ZAP-70 is critical for T cell activation
(45-47). ZAP-70 appears to be inactive in resting cells and, upon TCR
engagement, undergoes tyrosine phosphorylation and activation. In order
to determine whether the Crk-associated ZAP-70 is enzymatically active, we immunoprecipitated Crk, and ZAP-70 for comparison, from activated Jurkat T cells and performed an immune complex kinase assay
on the precipitate. A 70-kDa radiolabeled protein band was observed in
both ZAP-70 and Crk immunoprecipitates (Fig.
8A, upper panel), suggesting
the presence of catalytically active autophosphorylating ZAP-70 in the
Crk immunoprecipitate. Furthermore, the Crk co-immunoprecipitating kinase phosphorylated a ZAP-70-specific exogenous substrate, CFB3 (Fig.
8A, lower panel). Proteins that were pulled down from a lysate of activated Jurkat cells, using bead-immobilized GST-Crk-SH2 fusion protein, exhibited a similar phosphorylating activity (Fig. 8B), indicating that the Crk-SH2 domain-associated ZAP-70
was enzymatically active. As expected, the active ZAP-70 interacted with the GST-ZAP-70-SH2 domains, which served as a positive control. This was due to the ability of the tyrosine-phosphorylated
TCR-
-chain, which possesses three tandem binding sites for ZAP-70,
to interact simultaneously with the fusion protein and with Jurkat
cell-derived endogenous ZAP-70. In contrast, GST alone or GST-Grb2-SH2,
which cannot bind ZAP-70, did not exhibit phosphorylation of either the
70- or the 42-kDa (CFB3) protein bands (Fig. 8).
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Fig. 8.
The Crk-associated ZAP-70 from activated
Jurkat T cells is enzymatically active. Jurkat cells (4 × 107/group) were incubated with 1% pervanadate for 30 min
at 37 °C followed by lysis and incubation of the lysate with the
indicated protein A-agarose-bound Abs (A) or the indicated
bead-immobilized GST fusion proteins (B). After 1 h
incubation on a rotator at 4 °C, the beads were spun down, washed
three times in lysis buffer, and subjected to immune complex kinase
assay in the presence of [-32P]ATP, with (A,
lower panel, and B) or without (A, upper
panel) 1 µg of CFB3 as a substrate. Samples were boiled for 5 min and subjected to SDS-PAGE under reducing conditions, and
radioactive protein bands were visualized by autoradiography. Molecular
size markers (in kilodalton) are indicated on the right, and
the positions of radiolabeled protein bands corresponding to ZAP-70 and
CFB3 are indicated on the left. Results are representative
of three experiments.
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Predominant Association of ZAP-70 and Crk Occurs at the Membrane
Fraction--
The transient activation of ZAP-70, which follows
triggering of the TCR, is correlated with its translocation to the cell membrane and association with tyrosine-phosphorylated ITAM sequences on
the TCR--chain and CD3 subunits (4). The association of ZAP-70 with
Crk occurred only in activated T cells following phosphorylation of
ZAP-70 on tyrosine residues, suggesting that this event may also take
place at the level of the plasma membrane. We therefore fractionated
lysates of activated Jurkat T cells and determined the presence of
ZAP-70 in general, and tyrosine-phosphorylated ZAP-70 in particular, in
each fraction. We also tested the ability of these molecules to
associate with the GST-Crk-SH2 fusion protein. As previously, the
association of ZAP-70 with the Crk-SH2 domain was found to be dependent
on cell activation (Fig. 9). Furthermore, although similar levels of tyrosine-phosphorylated ZAP-70 were observed
in the cytosol and particulate fractions, association with the Crk-SH2
domain occurred predominantly at the particulate fraction.
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Fig. 9.
GST-Crk fusion proteins interact
predominantly with membrane fraction-residing tyrosine-phosphorylated
ZAP-70. Jurkat cells (4 × 107/group) were either
untreated or stimulated with 1% pervanadate
(perVO4) for 30 min at 37 °C, followed by
treatment with lysis buffer (A, lane 5) or fractionation
into cytosolic (cyt) and particulate (mem)
fractions (1st to 4th lanes). Cell lysate
supernatants were then incubated with 5 µg of GST-Crk-II-SH2
immobilized to glutathione-agarose beads (1st to 4th
lanes) or protein A-agarose-bound anti-ZAP-70 Abs
(5th lane). After 1 h of incubation on a
rotator at 4 °C, the beads were washed, bound proteins were eluted
and subjected to SDS-PAGE and immunoblotting with anti-ZAP-70 Abs
(A). Supernatants from the same cell fractions were also
used as a source for ZAP-70 immunoprecipitation followed by SDS-PAGE
and sequential immunoblotting with anti-ZAP-70 (B) and
anti-Tyr(P) Abs (C). Protein samples were either form 4 × 107 cell eq (1st to 4th lanes), or
2 × 105 cell eq (5th lane).
Molecular size markers (in kilodalton) are indicated on the
right, and the position of the ZAP-70 protein band is
indicated by an arrowhead. Results are representative of
three experiments.
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DISCUSSION |
Crk proteins have been implicated in signaling cascades that are
linked to various cell-surface receptors, including the antigen receptors on B (16-18) and T (19-22) lymphocytes. Engagement of the
BCR in RAMOS cells induced the association of tyrosine-phosphorylated Cas and Cbl with the Crk-SH2 domain, predominantly in the particulate fraction of activated B cells (16). In addition, Vav was inducibly associated with Crk in BCR-stimulated tonsilar B cells (17), and C3G,
which catalyzes guanosine triphosphate (GTP) exchange on Rap1 (17, 25),
constitutively associated with the Crk SH3 domain. Since C3G functions
as a negative regulator of Ras (48), it has been suggested that Crk
proteins function by down-regulating BCR-induced
Ras-dependent signaling pathways.
Analysis of T lymphocytes revealed that Crk proteins associate with Cbl
(19, 20) and Cas-L (49) in an activation-dependent manner
and with C3G in a constitutive manner (50). The latter study also
suggested that Rap1, which operates downstream of C3G, functions as a
negative regulator of the TCR-mediated IL-2 gene transcription and
thereby contributes to the maintenance of T cell anergy (50).
Our present findings demonstrate that T cell-derived Crk proteins
undergo cell activation-dependent association with the
ZAP-70 PTK. Association between the two molecules requires tyrosine
phosphorylation of ZAP-70 and is mediated by direct physical contact
between the Crk-SH2 domain and ZAP-70 phosphotyrosyl-containing
sequences. We also found that binding of ZAP-70 to adapter proteins in
T cells is selective and specific to Crk, but not Grb2, or Nck. This is
in contrast to other phosphoproteins, such as Cbl, that can interact
with Crk (20), Grb2 (51), and Nck (52) adapter proteins. Furthermore,
the Crk-associated ZAP-70 was found to be enzymatically active and,
apart from undergoing autophosphorylation, could phosphorylate a
ZAP-70-specific substrate, CFB3. The possibility that the
Crk-associated kinase reflects a PTK other than ZAP-70 cannot be
completely ruled out. However, depletion of ZAP-70 from a lysate of
activated Jurkat cells also removed the PTK that was pulled down by
GST-Crk (Fig. 2) and significantly reduced the phosphorylating activity
toward the 70- (ZAP-70) and 42-kDa (CFB3) protein bands (not shown).
The ZAP-70 PTK is a key regulator of signaling in T lymphocytes, and
its absence leads to a complete loss of the ability of T cells to
respond to antigenic stimuli (45-47). Activity of ZAP-70 is directly
regulated by the TCR which, upon engagement with a major
histocompatibility complex-bound peptide antigen, undergoes phosphorylation by tyrosine kinases. Phosphorylation occurs at ITAM
sequences in the cytoplasmic tails of the TCR -chain and CD3
subunits (53, 54). These phospho-ITAMs function as scaffolds for
catalytically inactive ZAP-70, and direct physical binding occurs by a
cooperative interaction of the ZAP-70 tandem SH2 domains with doubly
tyrosine-phosphorylated ITAMs (43, 55). Whereas TCR-bound ZAP-70 is
found in both agonist-responsive active T cells and
antagonist-responsive anergic T cells, ZAP-70 undergoes tyrosine
phosphorylation and activation only upon productive stimulation with an
agonistic peptide antigen (56, 57). Further studies demonstrated that
Lck is the putative ZAP-70-phosphorylating kinase (43) and that a
single tyrosine residue in the activation loop of the ZAP-70 kinase
domain (Tyr493) is critical for activation of the enzyme.
Therefore, phosphorylation of Tyr493 enables opening of the
activation loop of ZAP-70 which becomes more accessible to potential
cellular substrates (5, 6).
The findings that Crk association with ZAP-70 is
activation-dependent (Figs. 1, 3, and 4), together with the
observation by Iwashima et al. (43) that Lck is the upstream
regulator of ZAP-70, raised the question whether Lck is involved in the
regulation of ZAP-70 association with Crk. The analysis of the Jurkat
mutant subline, JCaM1, which is deficient in Lck, confirmed our
hypothesis and demonstrated that ZAP-70-Crk association occurs in
activated wild-type Jurkat cells but not in activated Lck-deficient
mutant cells (Fig. 5).
It is well established that T cell activation induces the association
of ZAP-70 with TCR pITAMs, but the fraction of the total ZAP-70
proteins associated with pITAMs and the subcellular distribution of
ZAP-70 in resting cells is still unclear. An overexpressed GFP-ZAP-70
was found to be diffusely distributed throughout the quiescent cell,
and it accumulated at the plasma membrane upon cell activation (58).
However, a large amount of ZAP-70 resided in the nucleus of quiescent
cells and, upon cell activation, underwent tyrosine phosphorylation
(58). In another study, the endogenous ZAP-70 was found to localize to
the cell cortex in a diffuse band and exhibit similar distribution
following TCR stimulation (59). This pattern of distribution was
dependent on the ZAP-70 kinase domain and not on the SH2-containing
regulatory region. Our studies demonstrate that about one-quarter of
the cellular ZAP-70 is found in the membrane fraction of non-activated
Jurkat cells and that a significant fraction of the cytosolic ZAP-70
translocate to the membrane fraction upon cell stimulation. Cell
activation resulted in tyrosine phosphorylation of ZAP-70 at both the
cytosolic and membrane fractions. However, the predominating
tyrosine-phosphorylated ZAP-70 that interacted with Crk-SH2 was
membrane-derived. The results suggest differences in the ability of
phosphotyrosyl residues of cytosolic and membrane-derived ZAP-70 to
interact with the Crk-SH2 domain. This may also reflect diversity in
the conformation of the molecules or differential association of ZAP-70
with other cell components. Alternatively, it is possible that despite
the fact that the phosphorylation levels of membranous and cytosolic ZAP-70 are similar, the sites of phosphorylation of ZAP-70 in the two
cellular fractions may differ, and only membranous ZAP-70 undergoes
phosphorylation at regions that function as binding sites for the
Crk-SH2 domain.
Recent findings demonstrated that the plasma membrane of cells contain
detergent-insoluble rafts enriched in glycolipids and phosphatidylinositol-anchored membrane proteins (60, 61). T cell
activation results in membrane compartmentalization and accumulation in
the rafts of activated TCR and associated signal-transducing molecules
(60, 62). The formation of activation-dependent clusters of
TCR-containing multimolecular complexes at the site of interaction of T
cells with the antigen-presenting cells has also been confirmed by
fluorescence digital imaging (3). It is interesting to note that this
process is followed by translocation of tyrosine-phosphorylated ZAP-70
to the rafts (59), questioning the possibility that the mechanism of
translocation may involve, or even require, the direct interaction of
phospho-ZAP-70 with Crk proteins.
Determination of sequence specificity of the peptide-binding sites of
various SH2 domains demonstrated that the Crk-SH2 prefers sequences
with a general motif pYXXP or, more specifically, pYDHP (where pY indicates phosphotyrosine) (63). Although an identical sequence has not been found in ZAP-70, the protein possesses three YXXP motifs, in which Tyr is at positions 221 (Y221CIP), 315 (Y315ESP), and 319 (Y319SDP) (64). Tyr315 is a putative
phosphorylation site on ZAP-70 in activated T cells, and the
Y315ESP region serves as an in vivo binding site
for the Vav SH2 domain (65). Although it is not yet known whether all
three tyrosine residues in the YXXP motifs undergo
phosphorylation in activated T cells, the current data suggest that Crk
interaction with ZAP-70 is mediated via one of these three
pYXXP motifs.
The findings that Crk functions as a positive regulator of apoptosis in
Xenopus eggs have indicated that the
v-crk-induced transformation of cells may not only reflect
interference with the normal growth-regulating signals but also
perturbation of signaling pathways that control cell death (10). The
complexity of the TCR enables the recognition and binding of specific
peptide antigens and sorting of the resulting signal into one of
several signaling pathways, including the apoptotic pathway. Although the role of Crk in T lymphocytes is still elusive, its association with
ZAP-70 which operates immediately downstream of the TCR suggests that
Crk may be involved in signaling pathways leading to T cell apoptosis,
especially after strong activation conditions, such as those evoked by pervanadate.
The association of ZAP-70 with Crk may serve additional functions in
activated T cells. Thus, simultaneous association of Crk with ZAP-70
and a ZAP-70-specific substrate will ensure high and efficient
phosphorylation of substrates, even those that occur at low abundance.
Studies of Src family members and Abl PTKs have shown that the SH2
domain of these PTKs preferentially bind phosphotyrosyl-containing sequences that are phosphorylated by their own catalytic domain (66,
67). This may lead to consecutive phosphorylation of the substrate
proteins. However, this does not apply to ZAP-70 in which the tandem
SH2 domains and the catalytic domain interact with completely different
sequences (41). Therefore, it is possible that ZAP-70 interaction with
Crk serves as a mechanism by which the Crk-SH3 domain selects and/or
restricts the putative ZAP-70 substrates which may co-cluster with and
become vulnerable to phosphorylation by ZAP-70.
It is also possible that interaction of a ZAP-70-bound Crk protein with
cytoskeletal elements via the SH3 domain, or other compartmentalized
components in the cell, will permit anchoring of ZAP-70 and increase
its concentration at selected subcellular locations where a critical
minimal number of PTK molecules is required for signaling. Finally,
another potential role for the interaction of tyrosine-phosphorylated
ZAP-70 with the Crk-SH2 domain may be maintaining the enzyme as a
phosphoprotein in its catalytically active state by protecting it from
dephosphorylation by protein tyrosine phosphatases.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of Microbiology
and Immunology, Faculty of Health Sciences, Ben Gurion University of
the Negev, P. O. Box 653, Beer Sheva 84105, Israel. Tel.:
972-7-647-7267; Fax: 972-7-647-7626; E-mail:
noah@bgumail.bgu.ac.il.
