By
§
From the * Department of Microbiology and Immunology, and the Department of Medicine and the § Howard Hughes Medical Institute, University of California, San Francisco, California 94143, and
Department of Molecular Genetics, Institute of Hepatic Research, Kansai Medical School, Moriguchi
570, Japan
Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor-mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav- ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor-mediated signal transduction.
Stimulation of the TCR and B cell antigen receptors
(BCR) initiates a cascade of signal transduction events
involving the activation of two families of protein tyrosine
kinases (PTKs), Src and Syk (1). The Src family members
initiate these events by phosphorylating the tyrosine residues within the immunoreceptor tyrosine-based activation
motifs (ITAMs) after TCR/BCR stimulation (1). The Syk
and ZAP-70 PTKs are subsequently recruited to the phosphorylated ITAMs, where they become phosphorylated
and activated (1). Activation of these kinases further leads
to tyrosine phosphorylation of numerous cellular proteins
including Vav, phospholipase C The protooncogene Vav is expressed exclusively in hematopoietic cells and contains an array of structural motifs,
including a guanine nucleotide exchange (GEF) domain for
the Rho/Rac/CDC42 family of small GTPases, a pleckstrin homology domain, and two src homology (SH) 3 domains that flank one SH2 domain (5, 6). Its homology to
Dbl and CDC24 and recent functional data in vitro and in
fibroblasts suggests that Vav functions as a GEF for the Rho/
Rac/CDC42 family of small GTPases (5). Vav plays a
critical role in lymphocyte development and activation,
since T and B cell numbers are severely reduced in the absence of Vav (9). The small numbers of T and B cells
which can develop in the absence of Vav display a profound
and specific defect in TCR- and BCR-mediated signal transduction. Moreover, overexpression of Vav in Jurkat T cells
results in a marked increase in basal nuclear factor of activated
T cells (NFAT) or IL-2 promoter-driven transcriptional activity, which is further enhanced by TCR stimulation
(12). However, the exact molecular mechanism by which
Vav functions in lymphocytes remains to be determined.
We were interested in identifying upstream kinase(s) responsible for Vav tyrosine phosphorylation. We have previously shown that the Vav SH2 domain is required for its
TCR/BCR-induced tyrosine phosphorylation (13). In addition, we and others have previously reported that tyrosine phosphorylated ZAP-70 can associate with the Vav
SH2 domain after TCR stimulation (13). Interestingly, both ZAP-70 (Y315) and Syk (Y348) contain a consensus
Vav SH2 domain binding sequence, YESP (16). By using
the chicken B cell DT-40 in transient transfection experiments, we show here that Y315 in ZAP-70 is critical for
antigen receptor-mediated signaling. We find that mutation of Y315 in ZAP-70 prevents its interaction with the
Vav SH2 domain. The point mutation in ZAP-70 also results in global defects in antigen receptor-mediated signaling events, as measured by the marked reduction in inducible tyrosine phosphorylation of ZAP-70, Vav, SLP-76,
and Shc. These data strongly suggest that Y315 of ZAP-70
plays a critical role in regulating ZAP-70 function.
DNA Constructs and Fusion Proteins.
The NFAT luciferase reporter construct was a gift from Dr. G. Crabtree (Stanford University, Stanford, CA). The Vav plasmid (pCI115) was constructed by subcloning human Vav into pCIneo (Invitrogen, San
Diego, CA). The parental plasmid for the ZAP-70 mutant was pCDNA3-ZAP-70. The Y315F mutant of ZAP-70 (ZAP70[Y315F]) was created by M13-based, oligonucleotide-directed,
site-specific mutagenesis procedures (17). The myc epitope-tagged,
wild-type ZAP-70 (pSXSRa-ZAP-myc) was provided by Dr. L. Samelson (National Institutes of Health, Bethesda, MD). DNA
encoding wild-type rat Syk was subcloned into the mammalian
expression vector pEFBOS. Glutathione S transferase (GST)-
VavSH2 was provided by Dr. S. Katzav (Israel Air Force Aeromedical Center, Tel Hashomer, Israel). The human Shc plasmid and the FLAG epitope-tagged human SLP-76 cDNA were provided by Dr. M. Gishizky (Sugen Inc., Redwood City, CA) and
Dr. G. Koretzky (University of Iowa, Iowa City, IA), respectively.
Antibodies and Peptide.
The mAb used for the stimulation of
the BCR was M4 (provided by Drs. M. Cooper and C.L. Chen,
University of Alabama, Birmingham, AL). Anti-Vav polyclonal
Ab was purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-ZAP-70 mAb (2F3.2) was described previously (18). The anti-myc epitope mAb
(9E10) was provided by Dr. J.M. Bishop. The peptide used in this
paper represents a biotinylated doubly phosphorylated version of
the second ITAM of the TCR Cell Lines, Transfections, and Luciferase Assays.
Wild-type and various mutants of DT-40 cells were maintained and transfected
transiently as previously described (17, 19). In brief, 30 µg of either an empty vector, wild-type ZAP-70 or ZAP-70(Y315F),
and 20 µg of NFAT-Luc construct was used. Cells were then
electroporated, processed, and assayed as described (17).
Immunoprecipitations, Protein Precipitations, Peptide Binding, and
Immunoblotting.
Cells were harvested, washed, were left either
unstimulated or stimulated with M4 (2 µg/ml), and then lysed as
previously described (13). Lysates were then immunoprecipitated
with the indicated antibodies. When precipitated with GST fusion proteins, lysates were first precleared with GST alone (10 µg) before they were precipitated with the indicated GST fusion
proteins (2-5 µg). Resulting immunoprecipitates or protein complexes were resolved by SDS-PAGE. Peptide binding and immunoblotting were carried out as previously described (18).
In Vitro Kinase Assay.
After transient transfection, wild-type
and mutant ZAP-70 were immunoprecipitated and in vitro kinase assays were performed as previously described (17). Samples
were then analyzed by SDS-PAGE, transferred to polyvinylidene
difluoride membrane, treated with 1 M KOH for 1 h, and then
subjected to autoradiography and immunoblotting.
To examine
functional requirements of Y315 in ZAP-70 in antigen receptor-mediated signal transduction, we transfected the Syk-deficient DT-40 B cells with either wild-type or the
mutated ZAP-70 (ZAP-70[Y315F]) along with a NFAT
reporter construct (19). Consistent with the previous reports (19, 20), loss of syk in DT-40 resulted in a complete
block in BCR-stimulated NFAT activation, a defect that
could be rescued by expression of wild-type ZAP-70 (Fig.
1). In contrast, mutation of Y315 in ZAP-70 markedly impaired its ability to reconstitute BCR-induced NFAT activation (Fig. 1).
To determine whether tyrosine 315 within
the YESP motif of ZAP-70 functions as the Vav binding
site, we transiently transfected Syk-deficient DT-40 cells
with either wild-type ZAP-70 or ZAP-70(Y315F) and examined their ability to interact with a GST fusion protein containing the Vav SH2 domain (GST-VavSH2). As shown
in Fig. 2 A, GST-VavSH2 fusion protein selectively bound
to wild-type ZAP-70 after BCR stimulation or by treatment with the protein tyrosine phosphatase inhibitor pervanadate (Fig. 2 A). In contrast, mutation of Y315 in ZAP70 markedly impaired its ability to bind to the Vav SH2
domain.
Lck also associates with ZAP-70 via its SH2 domain after
TCR stimulation (21, 22). Interestingly, both wild-type
ZAP-70 and ZAP-70(Y315F) from either BCR-stimulated
or pervanadate-treated lysates could bind efficiently to the
GST-LckSH2 domain (Fig. 2 B), indicating that Y315 in
ZAP-70 is specifically required for its interaction with the
Vav SH2 domain, but not Lck SH2 domain.
Although initial phosphopeptide mapping studies failed
to identify Y315 as one of the major tyrosine phosphorylated residues in ZAP-70, it is important to note that not all
of the phosphorylation sites observed by two-dimensional
peptide mapping were identified (23, 24). In fact, the corresponding residue (Y348) in Syk has been shown to be a
major in vitro autophosphorylation site and it serves as the
binding site for the Vav SH2 domain (15, 25). Moreover,
not only did mutation of Y315 in ZAP-70 abolish the Vav-
ZAP-70 interaction, this interaction could also be completely disrupted by the presence of a ZAP-70 peptide encompassing phosphorylated Y315 (14). These observations
strongly argue that Y315 in ZAP-70 does represent an in
vivo phosphorylation site after antigen receptor stimulation.
To assess whether the Y315 mutation affects ZAP-70-mediated Vav tyrosine phosphorylation, we transiently coexpressed
human Vav with empty vector, wild-type ZAP-70, ZAP70(Y315F), or wild-type Syk into Lyn and Syk doubledeficient DT-40 cells, in which BCR-induced Vav phosphorylation was completely absent (Fig. 3 A, data not shown,
and reference 26). Coexpression of Vav with either wildtype ZAP-70 or Syk, but not ZAP-70(Y315F), led to Vav
tyrosine phosphorylation, which was further induced by
BCR stimulation (Fig. 3 A).
To further examine the impact of Y315 mutation on
ZAP-70-mediated tyrosine phosphorylation of other downstream substrates, we analyzed the tyrosine phosphorylation
status of SLP-76 and Shc. Coexpression of wild-type ZAP70 with either SLP-76 or Shc in Syk-deficient or Lyn and
Syk double-deficient DT-40 cells resulted in BCR-stimulated SLP-76 or Shc phosphorylation (Fig. 3, B and C, and
data not shown). Surprisingly, mutation of Y315 in ZAP70 substantially impaired its ability to mediate phosphorylation of SLP-76 and Shc (Fig. 3, B and C, and data not
shown). In addition, mutation of Y315 also markedly reduced ZAP-70 tyrosine phosphorylation after antigen receptor stimulation in Syk-deficient cells and in Jurkat T
cells (Fig. 3 D and data not shown). Taken together, Y315
of ZAP-70 is not only required for Vav tyrosine phosphorylation, but also for tyrosine phosphorylation of other
downstream substrates such as SLP-76, Shc, and even for
ZAP-70 itself.
One explanation for the global defects of ZAP-70(Y315F) could be
that the Y315F mutation reduced ZAP-70 kinase activity.
Myc epitope-tagged ZAP-70 or ZAP-70(Y315F) was expressed in Lyn and Syk double-deficient cells and the kinase
activity of anti-myc epitope-tagged immunoprecipitates was
measured as both autophosphorylation and phosphorylation
of an exogenous substrate, band III. The in vitro kinase assay
failed to reveal a substantial difference between wild-type
ZAP-70 and ZAP-70(Y315F) in their abilities to phosphorylate band III, although there may be a modest reduction in
autophosphorylation of ZAP-70(Y315F) (Fig. 4 A).
Another critical step for ZAP-70 phosphorylation and
activation is binding of ZAP-70 to the ITAMs after receptor stimulation. We used a biotinylated doubly phosphorylated ITAM peptide to precipitate ZAP-70 from lysates of
Syk-deficient DT-40 cells transfected with either wild-type
ZAP-70 or ZAP-70(Y315F). Similar amounts of wild-type ZAP-70 and ZAP-70(Y315F) bound to the phosphorylated
peptide (Fig. 4 B). In addition, similar amounts of tyrosinephosphorylated TCR In summary, we demonstrate here that Y315 in ZAP-70
is required to interact with the Vav SH2 domain, and is
critical for ZAP-70-mediated gene activation. Notably, the
Y315-homologous residue in Syk is also required for its interaction with the Vav SH2 domain and for Vav phosphorylation (15). We provide evidence here that the Y315 mutation results in a global defect in ZAP-70-mediated signaling pathways, suggesting an important role of Y315
in regulating ZAP-70 function.
Antigen receptor stimulation results in the assembly of
multiprotein complexes, a process likely to facilitate efficient tyrosine phosphorylation and/or activation of appropriate signaling molecules (2). The Vav-ZAP-70 binding
via Y315 may be important in initiating the proper formation of such signaling complexes, as both proteins are able
to interact with many other signaling molecules (2). Since
Vav possesses a GEF domain for Rho/Rac/CDC42 (7, 8), the interaction between Vav and ZAP-70 may provide a
mechanism by which ZAP-70 activates downstream Rho/
Rac/CDC42-mediated signaling events such as cytoskeletal rearrangement. Mutation of Y315 in ZAP-70 may result
in an impaired recruitment, phosphorylation and/or activation of many proteins including ZAP-70, Vav, SLP-76, and Shc.
isoforms, Shc, and SLP76 (1). Tyrosine phosphorylation and/or activation of
these substrates ultimately results in downstream cytokine
gene induction and other effector functions.
chain (18).
Mutation of Y315 Impairs ZAP-70 Function.
Fig. 1.
Mutation of Y315 in ZAP-70 impairs its function in BCRmediated signal transduction. Syk-deficient DT-40 cells were transiently co-transfected with 20 µg of NFAT-Luc along with 30 µg of either an
empty vector, wild-type ZAP-70, or ZAP-70(Y315F). After transfection (24-40 h), cells were either left unstimulated or stimulated with either
anti-BCR (M4, 2 mg/ml) or PMA (50 ng/ml) plus ionomycin (1 µM)
for 6-8 h, and subsequently assayed for luciferase activity. The results are
shown as the fold induction of luciferase activity as compared with the activity in unstimulated cells transfected with the empty vector, which is
~200 arbitrary units. Luciferase activity was determined in triplicate in
each experimental condition. The data are representative of at least three
independent experiments. The lower panel represents anti-ZAP-70 blot
(mAb 2F3.2) of equivalent amount of lysates from different transfectants
in the luciferase assay described above.
[View Larger Version of this Image (29K GIF file)]
Fig. 2.
Y315 in ZAP-70 is required for its binding to the Vav SH2
domain. (A) Syk-deficient DT-40 cells were transiently transfected with
either an empty vector, wild-type ZAP-70, or ZAP-70(Y315F). Cells
were then either left unstimulated or stimulated with either anti-BCR or
pervanadate, and lysed. The lysates were first precleared with GST alone
and then mixed with a GST fusion protein containing the Vav SH2 domain. The protein complexes were blotted with anti-ZAP-70 mAb (2F3.2).
The lysate lanes represent approximately one tenth volume of the total
cell lysates used for GST precipitations. (B) Mutation of Y315 in ZAP-70
did not affect its ability to interact with the Lck SH2 domain. Syk-deficient cells were transfected as described in A and were either left unstimulated or stimulated with anti-BCR or pervanadate. The lysates were first
precleared with GST alone and then precipitated with GST fusion protein containing the Lck SH2 domain. The protein complexes were then
blotted with anti-ZAP-70 mAb (2F3.2).
[View Larger Versions of these Images (19 + 13K GIF file)]
Fig. 3.
(A) Vav is not phosphorylated in cells transfected with ZAP-70(Y315F). Lyn/Syk double-deficient DT-40 cells were transiently co-transfected with human Vav (pCI115) along with either an empty vector, wild-type ZAP-70, ZAP-70(Y315F), or Syk. After transfection (20-40 h), cells
were either left unstimulated or stimulated with anti-BCR (M4, 2 µg/ml) for 2 min, and then lysed. The lysates were immunoprecipitated with anti-Vav
polyclonal Ab and the immune complexes were blotted with antiphosphotyrosine Ab (4G10; top). The blot was then stripped and reblotted with antiVav polyclonal Ab (middle). Equivalent amount of lysates were taken from each experimental condition and resolved on a SDS-PAGE and then blotted with anti-ZAP-70 mAb (2F3.2; bottom). (B) Mutation of Y315 in ZAP-70 reduces ZAP-70-mediated SLP-76 tyrosine phosphorylation. Syk-deficient
DT-40 cells were transiently transfected with FLAG epitope-tagged human SLP-76 (FLAG-SLP-76) along with either an empty vector, wild-type ZAP70, or ZAP-70(Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated with anti-FLAG epitope antibody (M2)
and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with anti-FLAG antibody (bottom). Anti-ZAP-70 Western blot revealed equivalent expression between wild-type ZAP-70 and ZAP-70 (Y315F) (data not shown). (C) Mutation of Y315 in ZAP-70 also reduces ZAP-70-mediated Shc tyrosine phosphorylation. Lyn/Syk double-deficient DT-40 cells were transiently transfected with human Shc cDNA along with
either an empty vector, wild-type ZAP-70, or ZAP-70 (Y315F). Cells were stimulated and lysed as described in A. The lysates were immunoprecipitated
with anti-Shc mAb and the immune complexes were blotted with 4G10 (top). The blot was then stripped and reblotted with polyclonal anti-Shc antibody (bottom). Anti-ZAP-70 Western blot showed equivalent expression between wild-type ZAP-70 and ZAP-70(Y315F) (data not shown). (D) Mutation of Y315 reduces BCR-mediated ZAP-70 tyrosine phosphorylation. Syk-deficient DT-40 cells were transiently transfected with either a vector, a
myc epitope-tagged wild type or ZAP-70(Y315F). Cells were either left unstimulated or stimulated with anti-BCR or pervanadate for 2 min, and then lysed. The lysates were then immunoprecipitated with anti-myc antibody (9E10) and the immune complexes were blotted with 4G10 (top). The blot was
then stripped and reblotted with anti-ZAP-70 mAb (2F3.2) (bottom).
[View Larger Versions of these Images (24 + 21 + 21 + 23K GIF file)]
Fig. 4.
(A) Mutation of Y315 in ZAP-70 does not affect its intrinsic
tyrosine kinase activity. Lyn/Syk double-deficient DT-40 cells were transient transfected with either an empty vector, a myc epitope-tagged wildtype ZAP-70, or ZAP-70(Y315F). After transfection (24-40 h), the lysates were immunoprecipitated with anti-myc epitope (9E10) and subjected
to an in vitro kinase assay. The products were separated on a SDS-PAGE
gel and transferred to polyvinylidene difluoride membrane. The membrane was subjected to KOH treatment and then in vitro phosphorylated
proteins were detected by autoradiography (top). Expression of ZAP-70
was detected by immunoblotting the same membrane with anti-ZAP-70
mAb (2F3.2) (bottom). (B) Mutation of Y315 in ZAP-70 does not affect its
binding to receptor ITAMs. Syk-deficient cells were transfected with either an empty vector, ZAP-70, or ZAP-70(Y315F). After transfection
(24-40 h), the lysates were mixed with 1 µg of doubly phosphorylated
peptide encompassing the second ITAM of TCR chain, followed by
the addition of avidin beads to collect complexes. The complexes were
then blotted with anti-ZAP-70 mAb (2F3.2).
[View Larger Versions of these Images (25 + 18K GIF file)]
chains were found to co-immunoprecipitate with either form of ZAP-70 when analyzed in Jurkat
T cells (data not shown). Taken together, these data demonstrate that mutation of Y315 in ZAP-70 did not dramatically
affect its kinase activity or its binding to receptor ITAMs.
Address correspondence to Dr. Arthur Weiss, the Howard Hughes Medical Institute, Department of Medicine and of Microbiology and Immunology, University of California, 3rd and Parnassus Avenues, Box 0724, San Francisco, CA 94143.
Received for publication 14 March 1997.
Q. Zhao is an associate of and A.W. is an investigator of the Howard Hughes Medical Institute. This work was supported in part by a grant from the National Cancer Institutes (RO1 CA72531).We thank Dr. G. Crabtree for the NFAT reporter plasmid, Dr. D. Cantrell (Imperial Cancer Research Fund, London, U.K.) for the pEF-BOS expression plasmid, Dr. S. Katzav for the Vav cDNAs and GSTVavSH2 construct, Dr. M. Gishizky for the Shc cDNA, Dr. Koretzky for the FLAG-SLP-76, and Drs. N. van Oers, T. Finkel and D. Yablonski (University of California, San Francisco, CA) for critical reading of the manuscript and for helpful discussions.
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