Cbl promotes ubiquitination of the T cell receptor zeta through an adaptor function of Zap-70.

Triggering of the T cell antigen receptor (TCR).CD3 complex induces its ubiquitination. However, the molecular events that lead to ubiquitin conjugation to these cell surface molecules have not been defined. Here we report that Cbl, a RING-type E3 ubiquitin-protein ligase, promotes ubiquitination of TCR zeta chain, which requires its functional variant Src homology 2 domain and an intact RING finger. The tyrosine kinase Zap-70, which binds to both TCR zeta and Cbl, plays an adaptor role in these events. Mutations in TCR zeta, Zap-70, or Cbl that disrupt the interaction between TCR zeta and Zap-70 or between Zap-70 and Cbl reduce ubiquitination of TCR zeta. Our results suggest a novel mechanism by which Cbl negatively regulates T cell development and activation by inducing ubiquitination of the TCR.CD3 components.

T cell activation is initiated by the binding of antigenic peptides presented by major histocompatibility complexes (MHCs) 1 on the antigen presenting cells to the T cell receptor (TCR) (1,2). The TCR is assembled as a multiple subunit complex that consists of the specific antigen-binding TCR ␣ and ␤ heterodimer, signal-transducing invariant CD3␥, ␦, ⑀ chains, and the structurally distinct TCR chain. The signal capacity of the CD3 subunits and TCR chain is achieved through the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs), characterized by the presence of a pair of YXX(L/I) sequences separated by 6 -8 variable amino acids. Each CD3 subunit has one copy, and TCR chain has three copies of ITAMs. Engagement of the TCR induces rapid phosphorylation of tyrosine residues on the ITAMs through the activation of the Src family protein tyrosine kinases (PTKs) Lck and Fyn. The tyrosine phosphorylation of ITAMs in CD3 subunits and TCR chain creates the binding sites for the Src homology 2 (SH2) domains of the Syk family PTKs Syk and Zap-70, resulting in their subsequent recruitment and activation. The activated PTKs in turn propagate activation signals by phosphorylating multiple intracellular proteins, eventually leading to T cell activation. Of note is the fact that biochemical and genetic studies have documented a critical role for the TCR chain in transducing the signaling processes (3), which is further supported by the formation of TCR chain clustering in the T cell-antigen presenting cell interface during T cell recognition (4).
Engagement of the TCR by the peptide-MHC complex or anti-TCR⅐CD3 antibodies also results in down-regulation of the TCR⅐CD3 complex (5)(6)(7). Down-regulation of the TCR⅐CD3 complex is considered a self-limiting factor to terminate a sustained signaling in T cell-antigen presenting cell conjugates and is also proposed to be responsible for unresponsiveness and/or tolerance induction of the self-reactive T cells (8,9). Among all potential pathways leading to TCR⅐CD3 down-regulation, ubiquitin (Ub) conjugation to the TCR⅐CD3 subunits, and particularly to the TCR chain, has been shown in activated T cells (10,11). Ubiquitination of TCR chain occurs in response to TCR triggering, upon treatment with a phosphatase inhibitor, pervanadate, and is inhibited by the PTK inhibitor herbimycin (11). Multiple lysine residues in the intracellular domain of TCR chain are potential sites for Ub attachment (12). However, the molecular mechanism underlying TCR chain ubiquitination remains unclear.
Ubiquitination of a protein substrate occurs via a covalent isopeptide bond formation between the C-terminal glycine of Ub and the ⑀-amino group of lysine residues of the substrate and involves a cascade of enzymatic reactions of E1, E2, and E3 (13,14). Ub is activated first by an activating enzyme (E1) to form a high energy thiolester bond between Ub and E1 and is then transferred to a conjugating enzyme (E2). The E3s or Ubprotein ligases are the components responsible for specific substrate recognition and for promoting Ub ligation to the target protein. Therefore, the E3s can provide specificity to the Ub system.
Ubiquitination of cell surface receptors represents a general mechanism of turning off signal transduction induced by the ligand binding (15). Particularly, a family of receptor tyrosine kinases such as the receptors for epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor-1 go through ubiquitination upon receptor engagement, and this Ub conjugation process involves Cbl, an adaptor protein (16 -18). Cbl, a 120-kDa proto-oncogene product, was originally isolated as a cellular homologue of v-Cbl, a part of the transforming fusion protein of CAS NS-1 retrovirus causing leukemia in mice (19). Cbl consists of an N-terminal variant SH2 domain, a RING finger, and a C-terminal proline-rich domain with potential tyrosine phosphorylation sites. Indeed, genetic and biochemical studies have shown that Cbl family proteins including those from Drosophila and Caenorhabditis elegans attenuate intracellular signaling induced by the engagement of cell surface receptors (20). It is now understood that Cbl functions as an E3 Ub ligase with a RING finger that recruits a Ub-conjugating enzyme or E2 and an SH2 domain that recognizes activated receptor tyrosine kinases for Ub conjugation (18,(21)(22)(23).
Two genetic studies using Cbl gene-targeted mice showed that the cell surface expression of the TCR⅐CD3 complex is up-regulated in Cbl-deficient thymocytes (24,25). Cbl deficiency favors positive selection of thymocytes (25), suggesting an important role for Cbl in T cell development and activation. The fact that TCR chain is ubiquitinated and that Cbl acts as a RING-type E3 ligase for receptor tyrosine kinases prompted us to investigate whether Cbl also functions as an E3 for TCR chain to promote its ubiquitination. Here we show that Cbl indeed induces Ub conjugation to TCR chain, which requires a functional RING finger and the N-terminal variant SH2 domain of Cbl. Furthermore, mutations of TCR ITAMs reduce its ubiquitination, whereas coexpression of Zap-70 enhances the event. Significantly, it is shown that Zap-70 plays an adaptor role in Cbl-induced Ub conjugation to TCR chain. Our results provide a molecular mechanism by which Cbl regulates T cell function by promoting ubiquitination of TCR chain.
Plasmids-The human TCR chain cDNA was amplified by polymerase chain reaction using a human lymphocyte cDNA library (CLON-TECH) as the template and was subcloned into a pEF vector with an in-frame Myc epitope tag at the 3Ј-end. A truncated mutant with the second half of the second ITAM and the third ITAM removed (TCR 1-121) was amplified by polymerase chain reaction and subcloned into the pEF vector. Point mutations at the N-terminal tyrosine residue to phenylalanine at each of the three ITAMs in wild-type TCR (AYF, ABYF, ABCYF) or TCR 1-121 (AYF), or lysines 115 and 117 mutated to arginine in TCR 1-121 (TCR 1-121KR), were generated by sitedirected mutagenesis (QuickChange, Stratagene). The human Zap-70 cDNA was subcloned into pEF without or with an Xpress epitope. Point mutations at tyrosine 292 to phenylalanine in Zap-70 were made by site-directed mutagenesis. The HA epitope-tagged Ub cDNA has been described (26,27). The Cbl cDNAs encoding the full-length Cbl or Cbl G306E without or with an HA epitope in the pEF vector were described previously (28). A Cbl construct containing the N-terminal variant SH2 domain and the RING finger (SH2ϩRING, amino acids 1-481), or the RING finger and C-terminal proline-rich domain (RINGϩC, amino acids 355-906) was amplified by polymerase chain reaction and subcloned into pEFneo with an HA epitope at the 5Ј-end. Mutations at glutamic acid 369 to alanine (E369A), cysteine 381 to alanine (C381A), and tryptophan 408 to alanine (W408A) in the full-length Cbl or in Cbl 1-481 were generated by site-directed mutagenesis.
Cell Culture, Transfection, and Stimulation-Jurkat T cells or a Zap-70-deficient Jurkat T cell line (P116; kindly provided by Dr. Bob Abraham, Duke University, Durham, NC) were cultured in RPMI supplemented with 10% fetal bovine serum and antibiotics. For protein expression in Jurkat T cells, cells were transfected with the appropriate amount of plasmids (usually 1-10 g total) by electroporation (240 V, 960 microfarads; Bio-Rad). 293T kidney embryonic cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. For protein expression in 293T cells, cells were transfected with the appropriate amount of plasmids (usually 1-4 g total) by calcium precipitation. After 48 h, cells were collected, resuspended (2 ϫ 10 7 /ml) in 0.5 ml of medium, and treated with pervanadate, OKT3, or MG132 (50 M) for 30 min at 37°C. Cells were then pelleted and resuspended in 1ϫ Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM NaPiP, 2 mM Na 3 VO 4 , and 10 g/ml each aprotinin and leupeptin). Cells were lysed for 10 min at 4°C, and insoluble materials were removed by centrifugation at 15,000 ϫ g (4°C for 10 min). For displaying ubiquitinated protein bands, 0.1% SDS was added into lysis buffer to disrupt nonspecific protein-protein interaction.
Immunoprecipitation and Immunoblotting-For immunoprecipitation, lysates (ϳ1 ϫ 10 7 cells) were mixed with antibodies (1 g) for 2 h followed by the addition of 30 l of protein G-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for an additional 2 h at 4°C.
Immunoprecipitates were washed four times with 1ϫ Nonidet P-40 lysis buffer and boiled in 20 l of 2ϫ Laemmli's buffer. Samples were subjected to 8 or 10% SDS-polyacrylamide gel electrophoresis analysis, and electrotransferred onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with the indicated primary antibodies (usually 1 g/ml), followed by horseradish peroxidase-conjugated secondary antibodies. Membranes were then washed and visualized with enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech). When necessary, membranes were stripped by incubation in stripping buffer (62.5 mM Tris-HCl, pH 5.7, 100 mM 2-mercaptoethanol, 2% SDS) for 1 h at 70°C with constant agitation, washed, and then reprobed with other antibodies as indicated.

RESULTS
Cbl Induces Ub Conjugation to TCR Chain-Previous studies have shown that the TCR components including the TCR chain undergo activation-dependent ubiquitination (10,11). The present study was made to determine the molecular events underlying Ub conjugation to TCR. As the first step, we tried to establish an in vivo system in which TCR can be properly ubiquitinated. For this purpose, TCR chain tagged with a Myc epitope at the C terminus was expressed in Jurkat T cells in the absence or presence of HA-tagged Ub. The cells were left untreated or were treated with pervanadate, a phosphatase inhibitor, or with both pervanadate and MG132, a proteasome inhibitor, and the cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-HA. Coexpression of HA-Ub with TCR resulted in the formation of a slow migrating, higher molecular weight smear that was recognized by anti-HA antibody (Fig.  1a, top panel). Treatment of the cells with pervanadate increased Ub conjugation to TCR, and that was further enhanced by the presence of both pervanadate and MG132. To eliminate the interference of nonspecific co-precipitation of other proteins, we added 0.1% SDS in the lysis buffer. The same membrane was reprobed with anti-Myc antibody, showing equivalent amounts of TCR expression among all the samples (Fig. 1a, bottom panel).
The recent identification of Cbl as a RING-type E3 Ubprotein ligase (21) prompted us to examine whether Cbl can promote ubiquitination of TCR. Jurkat T cells were cotransfected with TCR and HA-Ub plasmids in the absence or presence of Cbl plasmid. Coexpression of Cbl with TCR significantly augmented Ub conjugation to TCR, which was further enhanced by pervanadate treatment (Fig. 1b). The results suggest that TCR can act as a substrate for Cbl E3 Ub ligase in T cells.
Previous studies have demonstrated that TCR cross-linking with OKT3 could also induce TCR ubiquitination (10,11). To examine whether Ub conjugation to TCR could be induced under OKT3-stimulated conditions in our system, T cells transfected with TCR and Cbl were left unstimulated or stimulated with OKT3 or pervanadate. OKT3 stimulation indeed augmented Ub conjugation to TCR, although to a lesser degree than with pervanadate stimulation (Fig. 1c). The data were consistent with previous publications (10,11), suggesting that TCR ubiquitination occurs under physiologic stimulation conditions.
Cbl RING Finger and the Linker Region Are Required for Its Ligase Activity-We have previously shown that mutation at cysteine 381 to alanine (C381A) of Cbl RING finger can disrupt the interaction of Cbl with Ubc E2 and ablate its ligase activity in an in vitro system (21). To examine the effect of Cbl C381A mutant on TCR ubiquitination, we coexpressed TCR with wild-type Cbl or Cbl C381A mutant. Consistent with our in vitro study, the mutation at cysteine 381 to alanine abrogated Cbl-induced Ub conjugation to TCR under both resting and pervanadate-stimulated conditions (Fig. 2a).
A recent crystal structure study on Cbl⅐UbcH7 complex showed that besides the Cbl RING finger, which contains the primary binding sites for Ubc E2, a link region between the N-terminal variant SH2 domain and the RING finger, also forms contact sites with UbcH7 (23). Of note, the glutamic acids 366 and 369 form intermolecular hydrogen bonds with UbcH7 arginine 15 and arginine 5, respectively. To investigate the role of this linker region in Cbl E3 ligase activity toward TCR ubiquitination, we generated a point mutation at glutamic acid 369 to alanine in Cbl and coexpressed with TCR. As compared with wild-type Cbl, the E369A mutant reduced its ability to induce TCR ubiquitination under both resting and pervanadate-stimulated conditions (Fig. 2b).
The RING fingers of Cbl family proteins contain a well conserved tryptophan residue (Trp-408), and mutation at this residue to alanine (W408A) reduces its binding to Ubc E2 and eliminates its ligase activity in vitro (21). To further confirm the importance of the Cbl RING finger in Ub conjugation to TCR, we also coexpressed the W408A mutant with TCR. In agreement with our in vitro data (21), the W408A mutant severely reduced TCR ubiquitination under resting and pervanadate-treated conditions (Fig. 2b). Taken together, the re-sults indicate that an intact RING finger and the linker region are required for Cbl to promote TCR ubiquitination.
Cbl N-terminal Variant SH2 Domain and the RING Finger Are Sufficient for TCR Ubiquitination-Cbl consists of an N-terminal variant SH2 domain, a RING finger, and C-terminal proline-rich sequences (20). We next examined the functional requirement of each domain in Cbl for the Ub conjugation to TCR. Truncated Cbl constructs containing Cbl variant SH2 domain and the RING finger (SH2ϩRING), or the RING finger and the C-terminal portion (RINGϩC) were generated and coexpressed with TCR. The SH2ϩRING mutant induced Ub conjugation to TCR to a similar degree as wild-type Cbl (Fig. 3a), under resting and pervanadate-stimulated conditions. However, the RINGϩC mutant showed markedly reduced ability to promote TCR ubiquitination, in comparison with either wild-type Cbl or the SH2ϩRING mutant. We then tested whether the RING finger mutation at cysteine 381 to alanine in the SH2ϩRING mutant has an effect on TCR ubiquitination. As compared with Cbl SH2ϩRING, the C381A mutant showed reduced ability in inducing Ub conjugation to TCR (Fig. 3b). The data collectively suggest that the N-terminal SH2 domain and the RING finger are primarily responsible for TCR ubiquitination.
Zap-70 Enhances Cbl-mediated Ub Conjugation to TCR-The E3 ligases are the components in the Ub system that mediate substrate recognition, in addition to their role in the recruitment of Ub-loaded E2, and help transfer Ub to the substrate and subsequent polyubiquitination (13,14). Because Cbl is not known to associate directly with TCR, an intermediate or adaptor molecule would be required for Cbl to form an  indirect complex with TCR and to transfer Ub to TCR. The importance of the Cbl N-terminal SH2 domain as demonstrated above may suggest that the putative adaptor molecule can interact with the variant SH2 domain and also with TCR. One of the potential molecules for this role is Zap-70, because it is the well characterized protein that binds to Cbl variant SH2 domian (20) and also to TCR (Zap-70, or chain-associated protein-70 as it was originally named) (1). We therefore tested the possibility of whether Zap-70 has any effect on TCR ubiquitination. Coexpression of Zap-70 with TCR enhanced Ub conjugation to TCR under resting conditions, and TCR ubiquitination was further enhanced by pervanadate stimulation (Fig. 4a).
We next assessed whether Zap-70 could further augment the Cbl-mediated effect on TCR ubiquitination. Ub conjugation to TCR was indeed increased by coexpression of Cbl and Zap-70 under both resting and pervanadate-stimulated conditions (Fig. 4b, lanes 4 and 10). Previous studies have showed that tyrosine 292 in Zap-70 is the binding site for the Cbl variant SH2 domain and that mutation at this site to phenylalanine can disrupt its interaction with Cbl (20). We then examined the effect of this mutation on TCR ubiquitination. As compared with wild-type Zap-70, Zap-70 Y292F mutant showed a reduced ability to cooperate with Cbl to induce Ub conjugation to TCR (Fig. 4b, lanes 6 and 12), although substantial level of TCR ubiquitination remained.
Previous studies have documented that a loss-of-function mutation in Cbl glycine 306 to glutamic acid (G306E) can also disrupt its interaction with Zap-70 (20). The effect of this mutation on TCR ubiquitination was assessed by coexpressing TCR with either the SH2ϩRING or its G306E mutant. The G306E mutant markedly lost its ability to induce TCR ubiquitination as compared with the SH2ϩRING (Fig. 4c).
To further confirm an adaptor's role for Zap-70 in Cbl-induced ubiquitination of TCR, we employed a Zap-70-deficient non-lymphoid 293T cells. The cells were transfected with plasmids containing HA-Ub, TCR, and Cbl in the absence or the presence of Zap-70 plasmid. Unlike in Jurkat T cells, Cbl alone induced Ub conjugation to TCR only slightly in 293T cells (Fig.  4d). However, coexpression of Cbl with Zap-70 markedly augmented ubiquitination of TCR. The data collectively suggest that Zap-70 can act as an adaptor for Cbl to induce Ub transfer to TCR.
Functional ITAMs Are Required for TCR Ubiquitination-The TCR⅐CD3 complex including TCR undergoes Ub conjugation in an activation-dependent manner (Refs. 10 and 11 and the present study). To further dissect the molecular events underlying Cbl-promoted ubiquitination of TCR, we generated a series of point mutations at the tyrosine residues of the three ITAMs on TCR and analyzed the effect of these mutations on TCR ubiquitination. Mutation at a single tyrosine residue to phenylalanine at the first ITAM (denoted as AYF) or a double mutation at the first two ITAMs (ABYF) affected Ub conjugation to TCR to a certain degree, although these effects were variable among different mutants (Fig. 5a). Significantly, a mutation with all of the three ITAMs mutated (ABCYF) showed a marked reduction in TCR ubiquitination under both resting and pervanadate-stimulated conditions. We then analyzed the effect of these mutations on the interaction with Zap-70. Jurkat T cells coexpressed with Zap-70 and the TCR mutants were left unstimulated or stimulated with pervanadate. The cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-Zap-70. Of all the TCR mutants, only the ABCYF mutant completely disrupted the binding to Zap-70 (Fig. 5b).
To further assess the role of TCR ITAMs in its ubiquitination, we examined the effect of the ABCYF mutant in the presence of Zap-70 or Cbl. The Zap-70-induced Ub conjugation to TCR was severely reduced in the ABCYF mutant (Fig. 5c). Similarly, the same mutation also reduced Cbl-mediated ubiquitination as compared with wild-type TCR (Fig. 5d). Collectively, the data suggest that any of three ITAMs on TCR can mediate Zap-70 interaction and the subsequent Cbl-mediated ubiquitination of TCR.
We then focused on the first ITAM of TCR to analyze its role in the Ub conjugation process. A previous study has shown that Ub conjugation to TCR chain occurs in multiple intracellular lysine residues (12). In the TCR 1-121 mutant, there are five lysine residues in the intracellular portion, whereas in the 1-121KR mutant, the last two lysine residues were mutated to arginines. We first examined whether the truncated mutants could be ubiquitinated. The 1-121 and the 1-121KR mutants displayed similar or slightly reduced Ub conjugation as compared with the full-length TCR under resting and pervanadate-stimulated conditions (Fig. 6a). Next, the effect of Cbl on their ubiquitination was analyzed by coexpressing Cbl and the full-length TCR or the truncated mutants. Under resting conditions, Cbl induced Ub conjugation to both the full-length TCR and the truncated mutants to a similar degree, although a slight reduction of ubiquitination in the truncated mutants was observed under pervanadate-stimulated conditions (Fig. 6b).
The role of the first ITAM on TCR was then analyzed by generating an AYF mutation in TCR 1-121 and by coexpressing them with Zap-70. The 1-121 AYF mutant displayed a significant reduction of Zap-70-induced Ub conjugation as compared with the unmutated TCR 1-121 under the same condi- tions (Fig. 6c). In agreement with this finding, the interaction of the 1-121 AYF mutant with Zap-70 was completely abrogated (Fig. 6d). Taken together, the results further support the notion that a single ITAM in TCR is sufficient to mediate its interaction with Zap-70 and its ubiquitination.
TCR, Zap-70, and Cbl Form a Macromolecular Complex-Previous studies have documented protein-protein interaction between TCR and Zap-70 and between Zap-70 and Cbl (1,2,20). However, it was not known whether TCR, Zap-70, and Cbl could form a single macromolecular complex. To address this possibility, we performed coimmunoprecipitation experiments in Jurkat T cells. Cell lysates from unstimulated or OKT3stimulated Jurkat T cells were immunoprecipitated with anti-Cbl antibody. The immunoprecipitates were blotted with anti-TCR antibody and then with anti-Zap-70 antibody. As shown in Fig. 7a, both TCR and Zap-70 were coimmunoprecipitated by anti-Cbl antibody in an activation-dependent manner. Similarly, the cell lysates were immunoprecipitated with anti-TCR, and the immunoprecipitates were blotted with anti-Cbl and then with anti-Zap-70 antibody. Both Cbl and Zap-70 were co-precipitated by anti-TCR antibody (Fig. 7b). The endogenous formation of TCR⅐Zap-70⅐Cbl complex in Jurkat T cells prompted us to reconstitute such interaction in transiently transfected T cells overexpressing Myc-TCR and HA-Cbl. As shown in Fig. 7c, anti-HA antibody precipitated Myc-tagged TCR and endogenous Zap-70 in an activation-dependent manner. However, the TCR ABCYF mutant was not precipitated under the same conditions. Equal portions of the same cell lysates were also incubated with anti-Myc antibody. Cbl and Zap-70 were precipitated in an activation-dependent manner from cells transfected with wild-type TCR but not with the ABCYF mutant (Fig. 7d).
To further confirm that Zap-70 plays an adaptor's role in Cbl⅐Zap-70⅐TCR complex formation, we examined whether such complexes could also be formed in Zap-70-deficient P116 T cells. Consistent with Fig. 7b, anti-Cbl antibody precipitated TCR and Zap-70 from normal Jurkat T cells in an activationdependent manner (Fig. 7e). However, under the same conditions, the amount of co-precipitated TCR was markedly reduced in P116 T cells, and no Zap-70 was detected in the same samples. The cell lysates from normal Jurkat T cells and P116 T cells were immunoblotted with anti-TCR, anti-Zap-70, and anti-Cbl antibodies. Equivalent amounts of TCR and Cbl were present in these two cell types, whereas Zap-70 was absent in P116 T cells (Fig. 7f). The results collectively suggest that TCR, Zap-70, and Cbl form macromolecular complexes in T cells, further supporting an adaptor's role for Zap-70 in Cblinduced Ub conjugation to TCR. DISCUSSION We and others have recently demonstrated that Cbl functions as an E3 Ub-protein ligase, with its RING finger recruiting Ub-loaded E2 conjugation enzymes and its variant SH2 domain binding to activated receptor tyrosine kinases for Ub conjugation (18,21,22). Numerous biochemical studies have documented an important role of Cbl in the immune system, particularly in T cell development and activation as a negative regulator (recently reviewed in Ref. 20). Consistent with this, Cbl gene-targeted mice display hyperplasia in the thymus, up-regulation of the expression of cell surface markers in thymocytes, and enhanced thymocyte activation (24,25). However, a molecular link between Cbl E3 Ub ligase activity and its negative regulation of T cell function remains to be elucidated. This study shows that Cbl acts as an E3 Ub ligase to promote ubiquitination of TCR chain, with the tyrosine kinase Zap-70 as an adaptor to bring together TCR⅐Zap-70⅐Cbl complex. Dis- ruption of this complex formation by mutations in the TCR ITAMs, in Zap-70, or in Cbl reduces Cbl-mediated Ub conjugation to TCR chain. The biochemical study is further supported by the increased expression of TCR in Cbl-deficient thymocytes (data not shown). Because Zap-70 also binds to the ITAMs of CD3 subunits (1, 2) and these subunits are ubiquitinated upon engagement of the TCR (10), our findings may infer a general mechanism by which Cbl negatively regulates T cell development and activation through its E3 Ub ligase activity toward ubiquitination of the TCR⅐CD3 components.
The identification of Cbl RING finger to recruit E2 Ub conjugation enzyme to allow transfer of Ub to the substrate is further supported by a recent crystal structure study showing Cbl RING finger⅐UbcH7 E2 complex (23). The Cbl RING finger consists of a three-stranded ␤-sheet, an ␣-helix, and two large loops. It provides a shallow groove formed by the ␣-helix and the two zinc-chelating loops into which the tips of UbcH7 loops pack. Consistent with this structure, mutations at Cys-381 of the RING finger can disrupt Ubc E2 interaction and ablate its ligase activity (21,22). Of note, in the RING finger⅐UbcH7 interface, trypophan 408 of the RING finger forms multiple contacts with the residues on UbcH7, suggesting an essential role of this residue in Ubc E2 binding. Indeed, we previously showed that mutation of this residue to alanine reduces its ability for E2 binding and its E3 activities in an in vitro ubiquitination system (21). Here we provided in vivo evidence for the critical role of cysteine 381 and tryptophan 408 of Cbl RING finger, because mutation at either of these two sites abrogates or reduces Cbl-mediated Ub conjugation to TCR chain.
The crystal structure study also identified a linker region that connects the Cbl N-terminal variant SH2 domain and the RING finger (23). Besides its key structural role in Cbl itself, the linker region forms intermolecular hydrogen bond contacts with UbcH7. It was suggested that the involvement of this linker region in Ubc E2 binding might explain the fact that certain RING-type E3s may need more peptide sequences than the RING finger for E2 binding (23). We found that mutation at glutamic acid 369, which forms contact with arginine 5 of UbcH7, to alanine reduced the ability of Cbl to promote TCR ubiquitination. The result may suggest that a direct contact between this linker region of Cbl and Ubc E2 is also required for its E3 ligase activity in vivo.
A functional role for this linker region in Cbl is supported by a previous observation that deletion of either tyrosine 368 or 371 renders Cbl oncogenic (29). Consistent with this observation, the mutation of tyrosine 371 to phenylalanine resulted in the inactivation of Cbl to promote ubiquitination of the epidermal growth factor receptor (18). It was therefore suggested that phosphorylation of this tyrosine residue could regulate the E3 ligase activity of Cbl (18). However, the crystal structure study showed that this tyrosine residue is in a buried environment not easily accessible for phosphorylation. Besides, it is not known at present whether this tyrosine residue is indeed phosphorylated and what could be the tyrosine kinase for the phosphorylation. This linker region more likely plays a structural role for Cbl to help keep Cbl variant SH2 domain and the RING finger, together with their binding proteins, in a proper conformation and/or orientation.
E3 ligases in the Ub system are responsible for recognition of the substrate and for the transfer of Ub to the substrate (13,14). The present study demonstrates that the tyrosine kinase Zap-70 plays a previously unappreciated adaptor role for Cbl to recognize TCR and to help transfer Ub to TCR. This notion is supported by the following findings: 1) ectopic expression of Zap-70 with Cbl enhances Cbl-induced TCR ubiquitination; 2) disruption of the interaction between TCR and Zap-70, or between Zap-70 and Cbl, reduces Cbl-induced Ub conjugation to TCR; 3) Cbl SH2ϩRING, which contains the Zap-70 binding site, is sufficient for TCR ubiquitination; and 4) TCR, Zap-70, and Cbl form macromolecular complexes in T cells. Therefore, in the TCR⅐Zap-70⅐Cbl complex, Zap-70 acts as a scaffold to bridge TCR, through an interaction of TCR ITAMs and the SH2 domains of Zap-70, to the vicinity of Cbl⅐Ubc E2 complex for Ub transfer, through an interaction of the negative regulatory tyrosine-292 in Zap-70 and the N-terminal variant SH2 domain of Cbl (30). Thus, the TCR⅐Zap-70 complex behaves like an intact receptor tyrosine kinase such as the epidermal growth factor receptor, which provides a phosphotyrosine site for the docking of Cbl variant SH2 domain and also provides lysine residues for Ub conjugation.
We found that even with the Zap-70 Y292F mutant or the Cbl G306E mutant, which have been shown to disrupt the interaction between Zap-70 and Cbl (20), the TCR ubiquitination was still augmented (Fig. 4, b and c). The result raises a possibility that adaptor molecules other than Zap-70 may also facilitate the process. To further support this notion, we found that TCR does form a weak complex with Cbl in Zap-70deficient P116 T cells (Fig. 7e). One candidate molecule in T cells could be the adaptor protein SLAP. Like Zap-70, SLAP was shown to interact with both TCR and the N-terminal variant SH2 domain of Cbl (31,32). Consistent with this idea, SLAP also negatively regulates TCR signaling (31). Obviously, further study is needed to examine whether SLAP can play an adaptor role similar to Zap-70 in Cbl-mediated TCR ubiquitination.
Analogous to the receptor tyrosine kinases, in which Cbl negatively regulates their function through ubiquitination and subsequent degradation (16,18), previous studies have documented a negative role of Cbl in the regulation of Syk/Zap-70 family kinases (33)(34)(35). Cbl was shown to inhibit Syk activity in basophilic cells (33) or to induce the down-regulation of the protein expression level of Syk (34) and Zap-70 (35) in COS-1 cells. The data may suggest that Cbl could directly promote Ub conjugation to Syk and/or Zap-70. However, under the same conditions in which Cbl enhanced TCR ubiquitination, it induced Ub conjugation to Zap-70 to a much lesser degree. 2 Although Cbl-induced inhibition of Zap-70 kinase activity and/or protein degradation could represent a direct role of Cbl on Zap-70, the present study supports an indirect model in which Zap-70 serves as an adaptor in Cbl-promoted ubiquitination of TCR chain. Should this be true, the enhanced activation of Zap-70 in Cbl-deficient thymocytes (24,25) may also reflect the up-regulation of TCR⅐CD3 expression on the cell surface (24,25) Lymphoid progenitors migrate from bone marrow to thymus and go through a series of defined stages of development to eventually generate peripheral T cell repertoire. During thymic development, these progenitors acquire various cell surface molecules such as TCR, CD4, and CD8. CD4 and CD8 double positive T cells then interact with the MHC expressed on antigen presenting cells to go through positive and negative selection processes (recently reviewed in Ref. 36). It is generally believed that the thymocytes receiving a moderate strength TCR signal are positively selected and develop further into mature T cells, whereas cells receiving signals that are either too weak or too strong will be eliminated by negative selection. By regulating the cell surface expression of TCR⅐CD3 components, Cbl may be involved in the fine tuning of the avidity and the strength of TCR/MHC interactions. Consistent with this, in Cbl-deficient mice, the MHC class II-restricted positive selection of CD4 ϩ T cells is enhanced (25). The present study provides a molecular link on Cbl-regulated thymocyte selection by promoting ubiquitination of TCR chain and, potentially, other TCR⅐CD3 components.