From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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ABSTRACT |
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Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events. However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood. In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively. Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl. However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl. This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl. These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.
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INTRODUCTION |
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Binding of antigenic peptides presented by major
histocompatibility complex molecules to the T cell receptor
(TCR)1-CD3 complex induces a
rapid increase in the activities of two families of nonreceptor PTKs,
i.e. the Src (Fyn and Lck), and Syk (Syk and Zap-70)
families (1, 2). Activation of Lck and/or Fyn leads to tyrosine
phosphorylation of immunoreceptor tyrosine-based activation motifs
present in the intracellular domains of CD3 and subunits, resulting
in the subsequent recruitment and activation of Zap-70 and Syk. The
activated PTKs in turn propagate activation signals by phosphorylating
multiple intracellular proteins, eventually leading to T cell
activation, lymphokine production, and proliferation. However, many
aspects of the underlying signaling cascades remain unclear.
The c-cbl (Casitas B-lineage lymphoma) protooncogene was originally isolated as a cellular homologue of v-cbl, a part of the transforming gene of the Cas NS-1 murine leukemia retrovirus (3, 4). The 120-kDa product of c-cbl, Cbl, is rapidly phosphorylated on tyrosine residues in response to stimulation of various cell surface receptors, including the TCR (5-7) (reviewed in Liu and Altman (5)), suggesting a critical role for Cbl in signal transduction pathways. The importance of Cbl in intracellular signal transduction is further emphasized by the observation that it associates with several other signaling molecules. Numerous studies have shown that Cbl binds constitutively to Grb2 (6-9). Tyrosine phosphorylation of Cbl results in its inducible association with phosphatidylinositol 3-kinase (7, 8, 10-12), with CrkL (13-20), and with Vav (21). We recently demonstrated an interaction between Cbl and 14-3-3 proteins (22), which depends on serine phosphorylation of Cbl (23).
Reconstitution studies in COS-1 cells showed that Zap-70 phosphorylates
Cbl in an Lck- and/or Fyn-dependent manner (24). Syk has
been shown to have a greater intrinsic enzymatic activity than Zap-70
in terms of Cbl tyrosine phosphorylation (25). Syk alone can cause
tyrosine phosphorylation of Cbl, which is enhanced by coexpression of
Lck (23). Overexpression of Syk in mast cells enhances the
FcRI-mediated tyrosine phosphorylation of Cbl (26). The notion that
Cbl is phosphorylated by Src and/or Syk family kinases is complemented
by the observations that Cbl can physically associate with these PTKs.
Both Src family kinases, such as Fyn (6, 8, 24, 27, 28) or Lyn (29),
and Syk (26, 27) or Zap-70 (24, 30), have been reported to associate
with Cbl. The association of Zap-70 with Cbl is
activation-dependent, and is mediated by a phosphotyrosine
(Tyr(P))-binding (PTB) domain in the N-terminal region of Cbl (30). A
point mutation (G306E) in this domain of c-Cbl, which corresponds to a
loss-of-function mutation in Sli-1, a Caenorhabditis
elegans Cbl homologue (31), disrupts the interaction of Cbl with
Zap-70, indicating that the N-terminal portion of Cbl is required for
the interaction. However, the C-terminal region of Cbl has also been
implicated in the constitutive interaction of Cbl with Syk in mast
cells (26).
The interactions of Syk/Zap-70 and Fyn with Cbl strongly suggest that Cbl is involved in Syk/Zap-70- and/or Fyn-dependent signaling pathways which are stimulated by TCR ligation. However, the regulation of Cbl tyrosine phosphorylation by upstream PTKs remains poorly understood. As a first step toward understanding the biological function of Cbl, we have undertaken a study of the interaction of Cbl with Syk and/or Fyn using genetic and biochemical approaches. We demonstrate that Syk binds to the N terminus of Cbl, whereas Fyn binds to its C terminus. We further define a critical tyrosine residue of Syk, Tyr-316, which is required for the direct interaction with, and for the tyrosine phosphorylation of, Cbl. In addition, our results suggest that Fyn can act as an adaptor protein that facilitates the interaction between Cbl and Syk and the tyrosine phsophorylation of Cbl, suggesting a coordinated regulation of Cbl tyrosine phosphorylation by Src and Syk family PTKs.
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MATERIALS AND METHODS |
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Antibodies--
Polyclonal rabbit anti-Cbl (c-15) and anti-Fyn
antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-Tyr(P) monoclonal antibody (mAb) 4G10 and anti-glutathione
S-transferase (GST) polyclonal antibody were from Upstate
Biotechnology (Lake Placid, NY). Anti-hemagglutinin (HA) mAb (12CA5)
was from Boehringer Mannheim. An anti-CD3 (OKT3) and an
anti-HIV-derived tag epitope (H902) (32) mAbs were purified from
ascites fluid by protein A-Sepharose affinity chromatography.
Horseradish peroxidase-conjugated F(ab')2 fragments of
donkey anti-rabbit IgG or sheep anti-mouse IgG were from Amersham
Corp.
Plasmids-- The pLex and pACT2 vectors (CLONTECH) were used in the yeast two-hybrid system. cDNAs corresponding to full-length Cbl, v-Cbl (Cbl residues 1-365), and the C-terminal portion of Cbl (Cbl-C; amino acid residues 450-906) were fused in-frame to the yeast Gal4 activation domain encoded by a pACT2 vector. The pLex vectors containing Zap-70, Syk, or Syk mutants including SykK395R, Y518F/Y519F, 1-298, 1-212, 213-628, Y341F, and Y345F, and the pACT2 vector containing Vav, were described previously (32). Additional Syk point mutations including Y316F or Y500F were made by site-directed mutagenesis based on the pLex-Syk vector. cDNAs encoding wild-type Syk or Syk Y316F were subcloned into a mammalian vector, pEFneo (33), which has been tagged with an HA epitope. An H902-tagged Syk in pME18S was described (32). Fyn cDNA in pME18S was provided by T. Kawakami. The Fyn cDNA derived from pME18S was subcloned into pLex in an in-frame fashion. cDNAs encoding HA-tagged wild-type Cbl, 70Z, v-Cbl, Cbl-C, and successive C-terminal deletion mutants of HA-tagged 70Z in pEFneo were reported (34). Cbl-C and its successive N-terminal deletion mutants constructed by using a polymerase chain reaction were subcloned into pEFneo downstream of an H902 tag epitope (32).
Yeast Two-hybrid System--
The yeast two-hybrid system used in
this study was essentially the same as described previously (32).
Briefly, the yeast strain L40 was cotransformed with pLex and pACT2
vectors containing appropriate cDNAs and cotransformants were first
screened on plates containing synthetic dropout (SD) medium lacking Leu
and Trp. Colonies were then picked up and grown on plates with SD
medium lacking Leu, Trp, and His to examine protein-protein
interactions. The transformants were also tested for -galactosidase
activity using a filter assay.
Cell Culture and Stimulation-- Simian virus 40 T antigen (TAg)-transfected human leukemic Jurkat T cells (Jurkat-TAg) were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. Cells were resuspended (2 × 107/ml) in 0.5 ml of medium, equilibrated at 37 °C for 5 min, and activated with OKT3 (4 µg/ml) for 5 min. Stimulation was terminated by adding 0.5 ml of 2× Nonidet P-40 lysis buffer (2% Nonidet P-40, 40 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 4 mM Na3VO4, 20 µg/ml each of aprotinin and leupeptin). Cells were lysed for 10 min at 4 °C and insoluble materials were removed by centrifugation at 15,000 × g (4 °C for 10 min).
COS-1 cells were cultured in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum at 37 °C in 5% CO2. For protein expression in Jurkat-TAg and COS-1 cells, cells were transfected with appropriate amount of plasmids (usually 10 µg total) by electroporation as described previously (23).Expression of GST Fusion Proteins in a Baculovirus System-- The cDNAs encoding v-Cbl, or Cbl-C were subcloned into a baculovirus GST fusion vector, pAc-GST (Pharmingen) which, in addition to encoding a GST fusion partner, also encodes a 6× His tag in an in-frame fashion. Recombinant virus carrying these Cbl constructs were generated by cotransfection with wild-type baculovirus DNA into the insect cell line, Sf9, using a Baculogold transfection kit (Pharmingen), were further amplified, and were used for infection of Sf9 cells. The GST-Cbl fusion proteins were then purified by using glutathione or Ni2+-nitrilotriacetic acid affinity columns.
In Vitro Binding Assay and Far Western Blotting-- For in vitro binding assays, cell lysates were incubated with 10 µg of GST fusion proteins for 2 h at 4 °C, followed by adding 40 µl of glutathione-Sepharose beads as described previously (35). After 1 h at 4 °C, the binding mixtures were washed extensively in 1× Nonidet P-40 lysis buffer and used for further analysis. The procedure for Far-Western blotting was described previously (23). The GST-Fyn SH2 fusion protein was purchased from Upstate Biotechnology.
Immunoprecipitation and Immunoblotting-- Lysates (1 × 107 cells) were mixed with antibodies for 2 h, followed by addition of 40 µl of protein A/G Plus-Sepharose beads (Santa Cruz Biotechnology) for an additional h at 4 °C. Immunoprecipitates (IPs) were washed four times with 1× Nonidet P-40 lysis buffer, and boiled in 30 µl 2× Laemmli's buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis, 10% gel, analysis and electrotransferred onto polyvinylidene difluoride membranes (Millipore). Membranes were immunoblotted with the indicated primary antibodies (usually 1 µg/ml), followed by horseradish peroxidase-conjugated secondary antibodies. Membranes were washed and visualized with enhanced chemiluminescence detection system (ECL, Amersham Corp.). When necessary, membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS for 1 h at 70 °C with constant agitation, washed, and then reprobed with other antibodies as indicated.
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RESULTS |
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The Interaction of Fyn with Cbl in Yeast-- It was previously reported that Fyn associates with Cbl in T cells, and that this interaction is increased upon T cell activation (28). To further define the interaction between Fyn and Cbl, we first used the yeast two-hybrid system. As shown in Fig. 1A, cells transformed with both full-length Cbl or Cbl-C (and Fyn), but not with v-Cbl, exhibited growth in medium lacking Leu, Trp and His, suggesting that the interaction of Cbl with Fyn is mediated by the C-terminal region of Cbl. As a negative control, no interaction was observed between Fyn and the empty pACT2 vector.
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The Interaction of Fyn with Cbl in Vitro and in Vivo-- To further examine the interaction between Fyn and Cbl, we performed in vitro binding assays using GST-Cbl fusion proteins. Cell lysates from unstimulated Jurkat T cells or from anti-CD3 (OKT3)-stimulated cells were incubated with GST alone, GST-Cbl-C, or GST-v-Cbl. The precipitates were then analyzed with an anti-Fyn antibody. As shown in Fig. 2A, Cbl-C, but not v-Cbl or GST alone, bound Fyn in an activation-dependent manner (top panel). As a control, the membrane was reprobed with an anti-Grb2 antibody, showing that Cbl-C, but not v-Cbl, constitutively binds Grb2 (bottom panel), as reported earlier (6-9).
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Analysis of the Syk-Cbl Interaction in Yeast-- Syk was recently found to interact with Cbl in rat basophilic leukemia cells (26) and in B cells (27). To further define the interaction between Cbl and Syk, we first used the yeast two-hybrid system. As shown in Fig. 3A, yeast cotransformed with Syk and v-Cbl, but not Cbl or Cbl-C, grew in SD medium lacking Leu, Trp, and His. No interaction was observed between Zap-70 and any of the three Cbl constructs. As a positive control, Syk, but not Zap-70, interacted with Vav (32). This result indicates that Cbl interacts with Syk in yeast via its N-terminal region.
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The Interaction of Syk with Cbl in Vitro and in Vivo-- To establish the physiological relevance of the Cbl-Syk interaction, it was further explored by an in vitro binding assay using GST-Cbl fusion proteins. Jurkat-TAg cells were transiently transfected with plasmids encoding HA-tagged wild-type or Y316F-mutated Syk. Lysates from unstimulated or anti-CD3-stimulated cells were incubated with GST alone, GST-v-Cbl, or GST-Cbl-C, and the precipitates were probed with an anti-HA antibody. As shown in Fig. 4A, GST-v-Cbl displayed activation-dependent interaction with wild-type Syk but not with Syk Y316F (lane 6 versus 8). A weak but consistent interaction was observed between GST-Cbl-C and wild-type Syk in OKT3-stimulated cells (lane 10), but not with Syk Y316 (lanes 11 and 12). The expression levels of Syk or Syk Y316F in the lysates were similar as detected by immunoblotting cell lysates with the anti-HA antibody (lanes 13 and 14). This result indicates that Tyr-316 of Syk is required for maximal interaction with Cbl. However, the weak interaction of Cbl-C with Syk suggests an alternative, additional mechanism independent of the Cbl N-terminal domain, which mediates (directly or indirectly) an association between Cbl and Syk.
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Regulation of Tyrosine Phosphorylation of Cbl by Fyn and Syk-- The interaction of Syk and Fyn with the N- or C-terminal regions of Cbl, respectively, suggested that these two PTKs coordinately regulate the tyrosine phosphorylation of Cbl. This is consistent with previous studies demonstrating that both Fyn and Syk can phosphorylate Cbl in different cells (23-26). Next, we addressed the role of Syk and Fyn in the tyrosine phosphorylation of Cbl. COS-1 cells were cotransfected with a Cbl plasmid and plasmids encoding wild-type Syk, Syk Y316F, or Fyn. When lysates from these cells were immunoblotted with an anti-Tyr(P) antibody, coexpressed Syk was found to induce tyrosine phosphorylation of Cbl (Fig. 5A, top panel; lane 2). However, the Syk Y316F mutant caused a significantly lower tyrosine phosphorylation of Cbl (lane 3). This did not reflect lower enzymatic activity of the Y316F mutant since this mutant was phosphorylated on tyrosine to at least the same extent as wild-type Syk (lane 3 versus 2). In contrast, coexpression of Fyn did not cause a detectable increase in the Tyr(P) content of Cbl as compared with cells transfected with Cbl alone (lane 4 versus 1). The same immunoblot revealed the proper expression of Syk, Syk Y316F, or Fyn in the transfected cells. Similar reduced phosphorylation of Cbl by Syk Y316F was observed when Cbl IPs from the cells were probed with an anti-Tyr(P) antibody (Fig. 5A, middle panel). Anti-Cbl immunoblot of Cbl IPs from the same cells revealed similar expression levels of Cbl in the different groups (bottom panel). These results suggest that the putative Cbl-binding site in Syk, Tyr-316, is required for optimal tyrosine phosphorylation of Cbl.
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Interaction between Fyn and Syk-- The above findings lend support for the notion that Fyn, via its association with Cbl, functions as an adaptor to facilitate the interaction between Syk and Cbl. Such an adaptor function would imply that Fyn also associates (directly or indirectly) with Syk. To address this, we analyzed potential interactions between Fyn and Syk both in vitro and in vivo. A GST-Fyn SH2 fusion protein directly bound to Syk as demonstrated by Far-Western blotting of cell lysates from Jurkat-TAg cells transfected with H902-tagged Syk or with an empty vector (pME18S) (Fig. 8A, top panel). Reprobing the same membrane with an anti-Tyr(P) antibody (middle panel) or with the H902 antibody (bottom panel) confirmed that the Fyn-SH2-binding protein comigrates with tyrosine-phosphorylated Syk.
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DISCUSSION |
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Cbl is rapidly phosphorylated on tyrosine upon cross-linking of the TCR·CD3 complex, and also associates with Fyn and the Syk family PTKs, Syk and Zap-70 (reviewed in Liu and Altman (5)). Understanding how the tyrosine phosphorylation of Cbl is regulated by these PTKs is critical for the elucidation of the biological function of Cbl in TCR-mediated signal transduction. Previous studies which have addressed the functional and physical interactions between Cbl and Fyn or Zap-70/Syk (5, 6, 24-28, 30) have, on occasion, led to apparently contradictory findings regarding the relative contribution of Fyn versus Zap-70/Syk to the tyrosine phosphorylation of Cbl or the role of distinct regions of Cbl in binding to these kinases. In the current study, we mapped the interactions between Cbl and Fyn or Syk by using genetic and biochemical approaches. We found that Syk and Fyn interact directly with the N-terminal and C-terminal regions of Cbl, respectively. Tyr-316 of Syk was required both for the interaction with Cbl and for its optimal tyrosine phosphorylation in activated T cells. In addition, evidence was provided that Fyn plays primarily an indirect role by interacting with both Cbl and Syk, and by enhancing Syk-mediated phosphorylation of Cbl. Taken together, these results suggest a coordinated mechanism through which Src- and Syk-family kinases interact to promote the phosphorylation of Cbl, thereby regulating its adaptor function.
Previous studies have shown that the N-terminal region of Cbl (or v-Cbl) interacts with Zap-70 in an activation-dependent manner (30). This implicated the existence of an N-terminal PTB-like domain in Cbl which binds to Tyr(P) residue(s) in Zap-70. The basis for the interaction of Syk with Cbl is less clear, however. Using the yeast two-hybrid system, we detected an interaction of the N-terminal region of Cbl with Syk (but not with Zap-70). The reported lack of interaction between Syk and the N-terminal region of Cbl (26) most likely stems from the fact that the non-interacting N-terminal construct used in that study (residues 1-480) includes the zinc Ring finger of Cbl, whereas our construct (v-Cbl; residues 1-365) does not. Presence of the Ring finger was shown to suppress the transforming activity of v-Cbl (36) and to inhibit its interaction with the activated epidermal growth factor receptor (37), and may similarly prevent the interaction between the PTB-like domain of Cbl and Syk.
The inability of Zap-70 to interact with Cbl in yeast reflects the fact that Zap-70 is inactive in this system (32). Indeed, the interaction between Syk and Cbl in yeast was dependent on intact catalytic activity and the kinase domain of Syk. Significantly, we demonstrated that mutation of Tyr-316 in Syk to phenylalanine disrupted its interaction with v-Cbl. This effect was specific since the same mutation did not affect the interaction between Syk and Vav. These findings were recapitulated in mammalian cells where Syk interacted with the N-terminal region of Cbl in an activation-dependent manner both in vitro and in vivo, and Tyr-316 of Syk was required for maximal association (Fig. 4) and optimal phosphorylation (Fig. 5) of Cbl. The most likely explanation for our findings is that Syk activates itself by autophosphorylation, leading to the phosphorylation of Tyr-316 (and other tyrosine residues) and subsequent binding to the N-terminal, PTB-like Cbl domain. Tyr-316 lies within a NPYEXX motif which is conserved in porcine, human, and murine Syk. This motif is similar to the NPXY motif which mediates binding to the PTB domain of several signaling proteins, e.g. Shc and IRS-1 (38).
Taken together, our findings suggest the following model to account for the functional and physical interactions of Cbl with Fyn and Syk/Zap-70. Phosphorylated (i.e. activated) Syk/Zap-70 interact directly with the PTB-like N-terminal region of Cbl, and represent the major kinases that mediate the phosphorylation of Cbl following TCR/CD3 ligation. While Fyn may phosphorylate Cbl to some extent, its primary function is that of an adaptor protein that associates with both Cbl and Syk/Zap-70. The association of Fyn with the C-terminal region of Cbl is mediated primarily by its SH3 domain in non-activated cells, and additional SH2-mediated binding to Tyr(P) residues may occur after activation. The additional Fyn-mediated indirect association of Syk with Cbl-C may stabilize the overall interaction between the two proteins and increase its affinity, and this event may be critical for the proper phosphorylation of Cbl needed to trigger biologically relevant downstream signaling events which are mediated by Cbl. Such a model can account for the apparent discrepancies regarding reported interactions between Cbl and Fyn or Syk/Zap-70, and is supported by our findings. Thus, Fyn and Syk/Zap-70 appear to play previously unappreciated and complex coordinated roles in mediating the tyrosine phosphorylation of Cbl.
The biological function of Cbl is still unclear. Several studies documented that mammalian Cbl or its C. elegans (Sli-1) and Drosophila (D-Cbl) homologues act as a negative regulators of PTK-mediated signaling pathways (31, 39-42). However, the mechanisms underlying the observed negative effects in different cell systems appear to be distinct. While the original finding in C. elegans suggested that Sli-1 is a negative regulator of Ras-dependent signals (31), other studies demonstrated that, in mammalian cells, Cbl is a negative regulator of the JAK-STAT (but not Ras) signaling pathway (41), or of Syk (39). The latter study also demonstrated that inhibition of Syk-mediated signals by Cbl depends on physical association between the two proteins, and that the proline-rich domain of Cbl was essential for this inhibition (39). However, another recent report demonstrated that a Cbl-mediated CrkL-C3G-Rap1 signaling pathway is responsible for the defect in IL-2 production in anergic T cells (42). It should be noted, however, that neither Sli-1 or D-Cbl, nor the Cbl region required for the inhibition of Syk, contains the CrkL-binding sites which are located in a distal C-terminal region of Cbl (16, 18). On the other hand, Cbl was also reported to be a positive regulator in a Src-mediated signaling pathway leading to bone resorption (43). Clearly, additional studies are needed to elucidate the role of Cbl in intracellular signaling pathways. The current findings, as well as future studies on the biological effects of Cbl mutants deficient in their interaction with PTKs and other binding proteins, provide a framework for understanding the functions of Cbl.
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FOOTNOTES |
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* This work was supported by United States Department of Health and Human Services Grants CA35299 and GM50819 (to A. A.).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: Division of Cell
Biology, La Jolla Institute for Allergy and Immunology, 10355 Science
Center Drive, San Diego, CA 92121. Tel: 619-678-4604; Fax:
619-558-3500; E-mail: yuncail{at}liai.org.
1 The abbreviations used are: TCR, T cell antigen receptor; GST, glutathione S-transferase; mAb, monoclonal antibody; PTK, protein tyrosine kinase; Tyr(P), phosphotyrosine; PTB, phosphotyrosine binding; IP, immunoprecipitate; HA, hemagglutinin; SD, synthetic dropout.
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REFERENCES |
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