From the Department of Microbiology and Immunology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140 and the § Department of Pathology, University of Western
Australia, Nedlands 6009, Australia
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ABSTRACT |
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Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in response to stimulation through the receptors for antigens, immunoglobulins, cytokines, and growth factors as well as through the integrins. Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction, since c-Cbl interacts with many crucial signaling molecules including protein-tyrosine kinases, adaptor proteins, and phosphatidylinositol 3'-kinase. Therefore, it is essential for our understanding of the functions of c-Cbl in signal transduction to identify its tyrosine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these sites in the interactions of c-Cbl with other signaling proteins. In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate treatment, as well as in response to TcR/CD3 ligation. Coexpression experiments in COS cells demonstrate that among T cell-expressed Src- and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk appear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T cells. Among these kinases, Fyn and Yes demonstrate strong binding to c-Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine-independent mechanisms.
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INTRODUCTION |
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c-Cbl is a protooncogenic protein initially identified as the cellular homologue of a transforming protein expressed by the murine Cas NS-1 retrovirus. The retroviral Gag-v-Cbl protein contains the 355 amino acid-long N-terminal domain of murine c-Cbl, which is 98.5% homologous to the N-terminal 357 amino acid residues of human c-Cbl. The sequence of c-Cbl is extended beyond the stop codon of v-Cbl and, unlike v-Cbl, features a long C-terminal domain containing a RING finger, a leucine zipper, and numerous proline-rich motifs. This C-terminal domain accounts for more than half the total length of c-Cbl, which equals 913 or 906 amino acids for murine and human proteins, respectively (1). Gag-v-Cbl and the truncated form of c-Cbl corresponding to its sequence in Gag-v-Cbl are present in both the nucleus and the cytoplasm, whereas c-Cbl is exclusively cytoplasmic (2). It has been demonstrated that c-Cbl undergoes tyrosine phosphorylation in response to the antigen receptor-mediated stimulation of T cells (3-5) and B cells (6-9), as well as in response to stimulation through other multichain immune recognition receptors (10, 11), cytokine and growth factor receptors (10, 12-19), and integrins (20, 21). Considering that Abl, the protein-tyrosine kinase (PTK)1 known to phosphorylate c-Cbl, fails to phosphorylate its viral form, it is likely that v-Cbl lacks tyrosine phosphorylation sites (22, 23). Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction in a variety of cell types, since c-Cbl interacts in an activation- and/or tyrosine phosphorylation-dependent manner with several signaling molecules including receptor and nonreceptor PTKs (4, 9, 11, 13, 15, 16, 24-26), adaptor proteins (9, 16, 19, 23, 27-35), phosphatidylinositol 3'-kinase (4, 5, 7-9, 16, 21, 32), and 14-3-3 proteins (36, 37). Because of the large number of interactions involving c-Cbl, it is crucial for our understanding of its functions to locate the tyrosine phosphorylation sites of this protein, to identify the PTKs phosphorylating these sites, and to determine the role of individual sites in the interactions of c-Cbl with other signaling proteins. These questions were earlier addressed in a study focusing on tyrosine phosphorylation of c-Cbl in Abl-transformed cells (23), in which two sites that are essential for the tyrosine phosphorylation of c-Cbl, Tyr700 and Tyr774, were identified. However, no information is available regarding the location of tyrosine phosphorylation sites in c-Cbl following external stimulation of T cells, the nature of the PTKs phosphorylating these sites, or the interactions of these phosphotyrosines with proteins that are involved in signal transduction in T cells.
In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate, a potent inhibitor of protein-tyrosine phosphatases, as well as to the TcR/CD3-induced stimulation. We also analyzed tyrosine phosphorylation of c-Cbl in COS cells coexpressing various Src- and Syk-related PTKs with either wild-type or mutant c-Cbl. This analysis demonstrates that Fyn, Yes, Src, and Syk are capable of phosphorylating c-Cbl. Expression of Fyn, Yes, and Syk is typical for T cells, although not restricted to them. Furthermore, Fyn and Syk are thought to play a major role in the TcR/CD3-mediated signal transduction (38-42). Fyn, Yes, and Syk phosphorylate the same tyrosine residues that are phosphorylated in stimulated T cells. Phosphorylation of c-Cbl by Lck was substantially less profound than that by other Src family PTKs. Zap, another PTK crucial for T cell activation, was unable to phosphorylate c-Cbl in the absence of other PTKs but phosphorylated it in the presence of Lck, albeit to a lower extent than Syk, Src, Fyn, or Yes. Furthermore, we have demonstrated that binding of c-Cbl to Fyn and Yes is higher than its binding to other T cell-expressed Src- and Syk-related PTKs. Physical interactions between Fyn and c-Cbl involve phosphotyrosine-dependent binding that is mediated by the Fyn SH2 domain. However, phosphotyrosine-independent mechanisms also contribute substantially to the overall binding of Fyn to c-Cbl. In contrast, binding of Yes and Src to c-Cbl is primarily phosphotyrosine-dependent.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- The cDNA of human wild-type c-Cbl and that of its C-terminal truncations containing 357 (v-Cbl), 480, and 655 (HUT) amino acids were described earlier (1, 2, 22, 43). These cDNAs were cloned into the pCEP4 vector for expression in mammalian cells. The pSV7d vectors expressing wild-type and kinase-inactive Zap (44, 45) were kindly provided by Dr. A. Weiss (University of California, San Francisco). The pSV7cII vectors expressing SykA and SykB as well as the kinase-inactive form of SykB (46) were kindly provided by Dr. B. Rowley (Bristol-Myers Squibb, Princeton, NJ). The pSGT/pSG5 vectors expressing Src family protein-tyrosine kinases and their enzymatically inactive forms (47, 48) were kindly provided by Dr. S. Courtneidge (Sugen, Redwood City, CA).
Mutagenesis--
The wild-type c-Cbl cDNA was cloned into
the pAlter Max vector (Promega, Madison, WI). Site-directed mutagenesis
was performed using 17-27-mer oligonucleotide primers homologous to
the putative tyrosine phosphorylation sites of c-Cbl according to the
manufacturer's recommendations. To introduce a Tyr Phe mutation
into the cDNA of c-Cbl, the second nucleotide of the Tyr-encoding
codon in each primer was changed from A to T. To restore tyrosine
sites, reverse mutations were introduced using the primers homologous
to the wild-type c-Cbl cDNA. All mutations were verified by DNA
sequencing in both directions. Amino acid sequences of the
corresponding fragment of wild-type c-Cbl and the mutant forms of c-Cbl
that were used in this study are shown in Fig.
1.
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Cells-- Jurkat leukemic T cells, their JCaM1.6 mutant lacking Lck, and COS-7 SV40-transformed green monkey fibroblast-like cells were obtained from ATCC. Jurkat and JCaM1.6 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), HEPES, L-glutamine, and antibiotics. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with the same reagents.
Transfection-- Plasmid DNA was transfected into Jurkat, JCaM1.6, and COS-7 cells using DMRIE-C lipid reagent (Life Technologies, Inc.) following the manufacturer's recommendations. 5 µg of cDNA was used per 2 × 106 T cells or 3 × 105 COS cells. pAlterMAX-based plasmids were used unless indicated otherwise. Cells were activated where indicated and lysed 24 h (Jurkat, JCaM) or 48 h (COS) after transfection.
Activation of Cells-- Jurkat cells were resuspended in RPMI 1640 supplemented with 20 mM HEPES at a density of 2 × 107/ml and activated by either pervanadate or the anti-CD3 mAb OKT3. Sodium vanadate and hydrogen peroxide were mixed in RPMI 1640 at final concentrations of 10 and 30 mM, respectively, preincubated for 10 min, and added to cell suspensions at a 1:100 dilution for an additional 10 min. OKT3 was added to the cells at a concentration of 2 µg/ml. F(ab')2 goat-anti-mouse IgG (Cappel, Durham, NC) at a concentration of 8 µg/ml was added 1 min later for an additional 3 min. JCaM1.6 and, where indicated, COS cells were activated with pervanadate as described above for Jurkat cells.
Antibodies and Fusion Proteins--
Antisera to Src family
kinases and Zap have been previously described (49, 50). The anti-Syk
antisera was kindly provided by Dr. J. Fargnoli (Bristol-Myers Squibb,
Princeton, NJ). The affinity-purified polyclonal antibody against c-Cbl
(C-15) and PY20 anti-Tyr(P) mAb (IgG2b) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). OKT3 anti-CD3 mAb was purified from
ascites fluid using protein A-Sepharose. Rabbit IgG against mouse IgG and goat F(ab')2 fragments against mouse IgG were purchased
from Cappel (Durham, NC). The anti-HA mAb 12CA5 (IgG2b) was kindly provided by Dr. T. Moran (Mt. Sinai Hospital, New York, NY).
Glutathione S-transferase fusion protein containing T
cell-specific Fyn SH2 domain was produced in Escherichia
coli using pGEX-2T expression vector and purified on
glutathione-Sepharose columns as described earlier (49).
Immunoprecipitation-- T cells (2 × 107/ml in serum-free medium) were lysed in TNE buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA, and 1% Nonidet P-40) supplemented with 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 1 mM sodium orthovanadate (Na3VO4) for 30 min on ice. Cell lysates were cleared from nuclei and debris by centrifugation at 14,000 × g for 5 min at 4 °C. Protein concentrations were determined in precleared lysates using Coomassie reagent (Pierce). Aliquots of these lysates containing equal amounts of total cellular protein were incubated with appropriate antisera or specific immunoglobulins for 1 h at 4 °C. Normal rabbit antisera or isotype-matched IgGs (Sigma) were added to some samples as specificity controls. This was followed by incubation with suspension of Staphylococcus aureus-derived PANSORBIN particles (Calbiochem) for an additional 1 h (25 µl of 10% suspension/1 µl of antiserum). When mouse antibodies were used for immunoprecipitation, Pansorbin particles were precoated with rabbit anti-mouse IgG. The immunoprecipitates were washed five times in TNE buffer and used for either immunoblotting or immune complex kinase assays.
Immune Complex Kinase Assays--
Immunoprecipitates were
additionally washed in kinase buffer (20 mM MOPS, pH 7.0, containing 5 mM of each MnCl2 and
MgCl2) and incubated for 5 min at room temperature in 25 µl of kinase buffer containing [-32P]ATP (NEN Life
Science Products) at a final concentration of 0.5 µCi/µl. The
reaction was stopped by boiling samples in 10 mM Tris
containing 1% SDS and 1 mM sodium orthovanadate. PANSORBIN particles were centrifuged at 14,000 × g for 2 min,
and supernatants were collected and diluted 1:10 with buffer containing
10 mM Tris, 50 mM NaCl, 5 mM EDTA,
1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10 mM sodium fluoride. Proteins of
interest were reprecipitated with the corresponding antibodies. These
immunoprecipitates were incubated with SDS-PAGE sample buffer for 10 min at room temperature and cleared of PANSORBIN by centrifugation.
Supernatants were boiled and subjected to SDS-PAGE separation.
Phosphoprotein bands were visualized by autoradiography. Reaction time
courses were examined for all kinases studied, and the 5-min point was determined to be in the linear range in each case.
Immunoblotting-- Immunoprecipitates were treated with SDS-PAGE sample buffer, and the recovered proteins were separated by SDS-PAGE and transferred to nitrocellulose (Bio-Rad). Nitrocellulose was blocked in Tris-buffered saline containing 1% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature and incubated with an appropriate antibody diluted in blocking buffer for an additional 1 h. For detection by autoradiography, blots were washed in Tris-buffered saline containing 0.1% Tween 20 and incubated with 1 µCi/ml of 125I-labeled protein A (ICN, Costa Mesa, CA) or 125I-labeled anti-mouse IgG (NEN Life Science Products) in blocking buffer for an additional 1 h. Bands were visualized using autoradiography unless indicated otherwise. For detection by enhanced chemiluminescence, blots were similarly treated with horseradish peroxidase-labeled antibodies to rabbit IgG and then incubated with the SuperSignal substrate system (Pierce). The intensity of protein bands was determined using Bio-Rad Scanning Densitometer model 1650 and found to be in the linear response range.
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RESULTS |
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Stimulation of T Cells Induces Phosphorylation of c-Cbl Tyrosines
700, 731, and 774--
Identification of tyrosine phosphorylation
sites in c-Cbl is complicated by the fact that 22 tyrosine residues are
present in the wild-type human c-Cbl. To focus on the location of
tyrosine phosphorylation sites, we expressed wild-type c-Cbl and a
C-terminally truncated form, termed HUT-Cbl, in Jurkat cells and
determined whether these recombinant proteins became
tyrosine-phosphorylated following activation of T cells with
pervanadate. The recombinant forms of Cbl were HA-tagged to facilitate
their separation from the endogenous c-Cbl. The full-length c-Cbl and
HUT-Cbl were immunoprecipitated with anti-HA, separated by SDS-PAGE and
immunoblotted using anti-HA and anti-Tyr(P) to assess their expression
and tyrosine phosphorylation, respectively. Although both proteins were
expressed at detectable levels, only the full-length c-Cbl protein
demonstrated phosphorylation on tyrosine under these conditions (Fig.
2). Likewise, two other truncated forms
of c-Cbl containing 357 and 480 amino acid residues exhibited no
phosphorylation in pervanadate-stimulated T cells (data not shown).
These findings indicate that the tyrosine-phosphorylated sites of human
c-Cbl are located between the C termini of HUT-Cbl (amino acid 655) and
wild-type c-Cbl (amino acid 906). This region includes tyrosine
residues 674, 700, 731, 735, 774, 869, and 871 of c-Cbl. Expression
vectors encoding mutant c-Cbl proteins, in which phenylalanines
were substituted for one or several of these tyrosines, were generated
using site-directed mutagenesis as described under "Experimental
Procedures" (see Fig. 1) and transfected into Jurkat cells. HA-tagged
c-Cbl mutant proteins were immunoprecipitated from lysates of
pervanadate-activated Jurkat cells, and their expression and tyrosine
phosphorylation were assessed using immunoblotting with anti-HA and
anti-Tyr(P), respectively. Anti-HA immunoblotting demonstrated that
expression levels for wild-type and mutant c-Cbl proteins were similar
in different samples (Fig. 3,
A and B), indicating that the anti-Tyr(P)
reactivity detected for these proteins correctly reflected their
tyrosine phosphorylation levels. Quantitation of the corresponding
bands demonstrated that tyrosine phosphorylation of the mutant c-Cbl
lacking tyrosines 674-7742 did not exceed 5%
of that for wild-type c-Cbl (Fig. 3, A and B). In
contrast, most of the single Tyr Phe mutations examined did not
show substantially decreased tyrosine phosphorylation of c-Cbl. The
only exception among the single mutations was Y774F, which detectably
reduced tyrosine phosphorylation of c-Cbl (Fig. 3A).
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Specificity of PTKs in Phosphorylation of c-Cbl--
To determine
which PTKs phosphorylate c-Cbl and to identify tyrosine residues of
c-Cbl that are phosphorylated by particular PTKs, we coexpressed
wild-type and Tyr5 Phe c-Cbl with Fyn, Yes, Src, Lck,
Zap, and Syk in COS cells and examined the levels of c-Cbl tyrosine
phosphorylation as described above for T cells. A major difference
between COS and T cells is that expression levels for transfected
cDNAs are considerably higher in COS than in T cells. Indeed, we
analyzed the expression levels of endogenous and recombinant c-Cbl in
COS cells and demonstrated that the amounts of both wild-type and
Tyr5
Phe c-Cbl in cells transfected with c-Cbl cDNA
substantially exceeded the endogenous level of c-Cbl in these cells
(Fig. 4A). This overexpression
might result in incomplete immunoprecipitation of the proteins
examined, introducing quantitative errors in the experimental results.
To address this issue, we subjected the lysates of COS cells
transfected with c-Cbl cDNA to three sequential rounds of anti-HA
immunoprecipitation. We then analyzed the amounts of c-Cbl in these
immunoprecipitates by anti-Cbl immunoblotting. The experiment
demonstrated that over 90% of HA-tagged c-Cbl was precipitated by
anti-HA in the first round (Fig. 4B). Overexpression of the
c-Cbl proteins might also increase their nonspecific binding to
immunoprecipitating antibodies. However, we found no precipitation of
wild-type or mutant c-Cbl with the isotype-matched nonspecific IgG2b
myeloma protein MOPC195 (Fig. 4C). Similar specificity
controls were used in all subsequent experiments and consistently
demonstrated the lack of detectable nonspecific immunoprecipitation.
Taken together, these results indicated that the conditions of
immunoprecipitation used in this study permitted comparing the amounts
of precipitated proteins in a quantitative fashion.
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Role of Tyrosine Phosphorylation in the Association between c-Cbl and PTKs-- We also examined the binding of c-Cbl to PTKs in COS cells by immunoprecipitation of either c-Cbl or the corresponding PTKs followed by immunoblotting with anti-PTKs and anti-HA, respectively. The choice of this experimental system was determined, among other things, by the insufficient coimmunoprecipitation of c-Cbl with PTKs from Jurkat T cells (data not shown). This finding was consistent with our earlier reports revealing only a modest binding between c-Cbl and Fyn in vivo and in vitro (26). The results shown in Fig. 8A demonstrated that Src, Fyn, and Yes were able to coprecipitate with wild-type c-Cbl, albeit at different levels. In contrast, no substantial binding to c-Cbl was observed in this system for Lck, Zap, and Syk (Fig. 8A and data not shown). These results indicated that binding of Src family PTKs to wild-type c-Cbl correlates with their ability to phosphorylate this protein, which appears to be higher for Src, Fyn, and Yes than for Lck (see Fig. 7).
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DISCUSSION |
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It has previously been shown that several proteins, including
PTKs, interact with c-Cbl in a tyrosine
phosphorylation-dependent manner and that several PTKs of T
cells are capable of phosphorylating c-Cbl. In this report, we identify
the tyrosine phosphorylation sites of c-Cbl in stimulated T cells as
tyrosines 700, 731, and 774. This identification is based on the
analysis of tyrosine phosphorylation of the c-Cbl molecules containing
Tyr Phe mutations, as well as on the analysis of Phe
Tyr
reconstitution mutations of the tyrosine phosphorylation-defective
Tyr5
Phe form of c-Cbl, which lacks tyrosines 674, 700, 731, 735, and 774. Most of our experiments were conducted with
reconstitution mutations, because a decrease in tyrosine
phosphorylation caused by a single Tyr
Phe mutation was typically
more difficult to detect than an increase caused by a single
reconstitution Phe
Tyr mutation. This fact can be explained by low
partial contributions of some tyrosine phosphorylation sites to the
overall phosphorylation of c-Cbl, and/or by an increase in
phosphorylation of secondary sites followed by the removal of any major
phosphorylation site. Tyrosines of c-Cbl other than 700, 731, and 774 do not appear to be substantially phosphorylated in T cells. First of
all, the mutant form of c-Cbl, which lacks tyrosines 700, 731, and 774 is not appreciably phosphorylated. Furthermore, the reconstitution mutations of tyrosines 674 (F674Y) and 735 (F735Y) do not result in a
detectable increase in c-Cbl tyrosine phosphorylation. Finally, the
level of tyrosine phosphorylation for the c-Cbl proteins containing double and triple reconstitution mutations approaches that of the
wild-type c-Cbl. However, the possibility cannot be ruled out entirely
that mutations of c-Cbl induce structural changes in this protein,
preventing recognition and/or phosphorylation of other sites of c-Cbl
by PTKs. In such a case, some of the phosphorylation sites might go
undetermined using this approach. Nevertheless, this possibility does
not invalidate the identification of tyrosines 700, 731, and 774 as
notable tyrosine phosphorylation sites of c-Cbl.
Although the pervanadate-induced tyrosine phosphorylation is caused by the inhibition of protein tyrosine phosphatases and not by the activation of PTKs as in the case of TcR/CD3 ligation (51), the same sites of c-Cbl are phosphorylated following both pervanadate treatment and TcR/CD3 ligation. Moreover, the relative phosphate acceptor potentials of c-Cbl tyrosine phosphorylation sites appear to rank in the same order for both anti-CD3 and pervanadate stimulation of Jurkat cells. These findings are consistent with the fact that pervanadate mimics natural stimuli with respect to both early signal transduction events and biological responses in T cells (51, 53, 54) as well as in other cell types (55-59).
The results obtained by the expression of wild-type and mutant forms of c-Cbl in Jurkat cells do not allow us to determine what PTKs phosphorylate c-Cbl. It was previously demonstrated that Fyn (25, 26, 49, 60), Syk (24, 61, 62), and Zap plus a Src family PTK (25) are capable of phosphorylating c-Cbl. Therefore, we further analyzed the ability of four Src family kinases, Fyn, Src, Yes, and Lck, as well as both Syk-family kinases, Zap and Syk, to phosphorylate c-Cbl in COS cells. Among Src family PTKs, Src, Fyn, and Yes phosphorylate c-Cbl to a high level, whereas Lck phosphorylates c-Cbl rather poorly. It is impossible to explain the observed differences in the ability of various Src family PTKs to phosphorylate c-Cbl by their differential enzymatic activities toward all kinds of substrates. Indeed, the enzymatic activity of Lck in COS cells, as judged by its autophosphorylation, is substantially higher than that of Yes, approaching that of Fyn. Despite the relatively high autokinase activity of Lck, the level of c-Cbl tyrosine phosphorylation in Lck-expressing COS cells barely exceeds such a level in COS cells transduced with an empty vector. This result is consistent with the ability of Fyn and Yes, but not Lck, to coprecipitate with c-Cbl from T cell lysates and to phosphorylate c-Cbl in these immune complexes (25, 26, 49). The ability of Src-related PTKs to phosphorylate c-Cbl correlates with their structure, because Src, Yes, and Fyn belong to the same structural subfamily of Src-related PTKs, whereas Lck is substantially different from the first three kinases (63).
Among Syk family PTKs, only Syk, and not Zap, is capable of
phosphorylating c-Cbl in the absence of Src family PTKs. Interestingly, the difference between these two Syk family PTKs with regard to their
ability to phosphorylate c-Cbl closely mirrors their differences regarding the tyrosine phosphorylation of the chain of the TcR-CD3 complex (64). Taken together, these results suggest that Fyn and Syk
play a major role in tyrosine phosphorylation of c-Cbl in stimulated T
cells. Although Yes is also capable of phosphorylating c-Cbl when
coexpressed in COS cells, its role in the activation-induced tyrosine
phosphorylation of c-Cbl in T cells is unclear, because the
physiological stimuli that regulate the activity of Yes in T cells
remain to be defined. Therefore, c-Cbl appears to be preferentially phosphorylated by PTKs that are expressed ubiquitously, not in a T
cell-specific fashion. In conjunction with the fact that c-Cbl itself
is expressed in a wide variety of cell types and becomes tyrosine-phosphorylated in response to many distinct stimuli, this
finding suggests that c-Cbl-dependent signaling is not cell type-specific but is, instead, ubiquitous.
Interestingly, all PTKs that have been shown to phosphorylate c-Cbl in this study phosphorylate the same three sites of this protein, namely tyrosines 700, 731, and 774. These are the same residues that are phosphorylated in response to T cell activation. Indeed, each of the phosphorylated tyrosines is surrounded by a sequence containing recognition elements for Src as defined earlier in Ref. 65. Most of these recognition elements are negatively charged amino acid residues (see Fig. 1), which are a characteristic feature of many tyrosine phosphorylation and autophosphorylation sites (66). Importantly, none of the tyrosine phosphorylation sites of c-Cbl exhibits recognition elements for Lck (65), consistent with the actual ineffectiveness of Lck in tyrosine phosphorylation of c-Cbl in COS cells. It should be noted, however, that the search of the c-Cbl amino acid sequence for potential tyrosine phosphorylation sites as defined in Ref. 65 suggests that tyrosines 141, 368, 371, and 455 also are sound candidates for being phosphorylated by Src. Our results demonstrate no appreciable phosphorylation of these residues, indicating that the actual tyrosine phosphorylation sites of a large protein molecule, such as c-Cbl, might differ from those predicted.
These experiments also indicate that the double and the triple
reconstitution mutations of the Tyr5 Phe c-Cbl result
in higher tyrosine phosphorylation levels for the "reconstituted" c-Cbl proteins than the simple sums of tyrosine phosphorylation for the
corresponding single reconstitution mutations (see Figs. 3 and 5-7).
It should be noted that all PTKs phosphorylating c-Cbl demonstrate this
trait, although it is more evident for Yes and Syk (Figs. 6 and 7).
These results argue that there is a certain degree of positive
cooperativity between the tyrosine phosphorylation sites of c-Cbl,
which may be due to the fact that phosphorylation of the first site
generates a "docking spot" for PTKs facilitating further
phosphorylation of c-Cbl.
To determine the role of tyrosine phosphorylation in physical interactions of c-Cbl with Src family and Syk family PTKs, we examined the association of wild-type and mutant forms of c-Cbl with these PTKs. The results of these experiments indicate that association of Src family PTKs with c-Cbl is usually stronger than that of Syk family PTKs, although substantial variations are evident within the Src family. Both phosphotyrosine-dependent and -independent mechanisms are involved in these interactions, although their relative contributions to the overall binding appear to be different for various PTKs. Thus, the role of tyrosine phosphorylation is substantial, but not predominant, for Fyn, whereas Yes and Src bind to c-Cbl primarily through the phosphotyrosine-dependent interactions. Consistent with this notion, Yes is capable of binding to any form of c-Cbl containing at least one tyrosine phosphorylation site. In contrast, phosphotyrosine-dependent binding of Fyn and its SH2 domain to the c-Cbl molecules containing only a single reconstitution mutation is very weak and is dramatically increased by reconstitution of additional tyrosine phosphorylation sites. This synergism between tyrosine phosphorylation sites of c-Cbl may be due to the fact that, for some PTKs, several PTK molecules have to be associated with the molecule of c-Cbl to stabilize this complex.
The tyrosine phosphorylation of c-Cbl is likely to have multiple consequences for signal transduction and cell activation. First of all, phosphorylation of one site may facilitate further phosphorylation of c-Cbl as a result of Src family PTK binding to this site. Furthermore, the existence of several tyrosine phosphorylation sites allows simultaneous binding of several signaling proteins to c-Cbl in an activation-dependent manner. In turn, simultaneous binding of PTKs and their substrates to c-Cbl might result in tyrosine phosphorylation of these substrates. It has earlier been proposed that a similar mechanism might promote Fyn-driven phosphorylation of Zap, a small fraction of which was found to be associated with c-Cbl (25). Furthermore, interactions between many other proteins might also be induced by their simultaneous binding to c-Cbl. Indeed, Tyr(P)-dependent binding of c-Cbl to various signal transduction proteins appears to be a hallmark of receptor-mediated activation in many types of cells. Thus, phosphorylation of tyrosines 700 and 774 has been shown to mediate binding of c-Cbl to the SH2 domain of CrkL in Abl-transformed cells (23). The interaction of c-Cbl with CrkL and Crk has earlier been detected in TcR/CD3-stimulated T cells (27, 29, 30), as well as in Bcr/Abl-transformed cells (32-34). The Crk family adaptor proteins are involved in the activation of small GTP-binding proteins through binding to the GTP/GDP exchange factors SOS and C3G (67-71). Indeed, it was recently reported that tyrosine phosphorylation of c-Cbl in T cells is linked to the activation of Rap1, a small GTP-binding protein regulated by C3G, which appears to down-regulate the TcR/CD3-mediated transcription of the interleukin-2 gene (72). Furthermore, tyrosine 731 is part of the c-Cbl sequence (Y731EAM) that has been reported to be responsible for the activation-dependent binding of phosphatidylinositol 3'-kinase to c-Cbl (73). Therefore, phosphorylation of tyrosine 731 is likely to be critical for the formation of the phosphatidylinositol 3'-kinase-c-Cbl complex, which has been detected in a variety of cell types following stimulation (4, 5, 7-9, 16, 21, 74), as well as in transformed cells (32). In addition, it has been reported (75) that tyrosine 700 might mediate binding of c-Cbl to Vav, a protein that plays a very important role in signal transduction in hematopoietic cells (76, 77). In summary, the data presented here demonstrate that TcR/CD3 ligation triggers phosphorylation of c-Cbl tyrosines 700, 731, and 774, all of which appear to be involved in the interactions of c-Cbl with crucial components of T cell signal transduction pathways, such as PTKs, phosphatidylinositol 3'-kinase, CrkL, and Vav. Hence, phosphorylation of these c-Cbl tyrosine residues is likely to play a crucial role in biologic responses of T cells and, possibly, other cell types to external stimuli.
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ACKNOWLEDGEMENTS |
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We thank S. Courtneidge, J. Fargnoli, T. Moran, B. Rowley, and A. Weiss for providing reagents; S. Gordon for technical assistance; R. Penhallow and P. Salgame for critical reading of the manuscript; and G. Harvey for editorial help.
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FOOTNOTES |
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* This work was funded by Council for Tobacco Research Award SA048 (to A. Y. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Kresge Bldg., Room 506, Dept. of Microbiology and Immunology, Temple University School of Medicine, 3400 Broad St., Philadelphia, PA 19140. Tel.: 215-707-1745; E-mail: tsygan{at}astro.ocis.temple.edu.
1 The abbreviations used are: PTK, protein-tyrosine kinase; HA, hemagglutinin tag; mAb, monoclonal antibody; Tyr(P), phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; TcR, T cell antigen receptor; SH2, Src homology 2; MOPS, 4-morpholinepropanesulfonic acid.
2
Denoted in the figures as 5YF and in the text
as Tyr5
Phe.
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REFERENCES |
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