Negative Regulation of T Cell Antigen Receptor-mediated Crk-L-C3G Signaling and Cell Adhesion by Cbl-b*

It was previously reported that Cbl-b associates with Crk-L in Jurkat T cells. However, the physiological significance of such association remains unclear. Here we examined a regulatory role of Cbl-b in Crk-L-C3G signaling pathway. We found that Cbl-b associates with, and induces, ubiquitin conjugation to Crk-L, which requires a functional RING finger. Cbl-b deficiency does not affect Crk-L stability, but its association with C3G. In Cbl-b–/– T cells, the interaction between Crk-L and C3G, and the activity of the small GTPase Rap1, are increased. Cbl-b–/– T cells also display increased adhesion and cell surface binding to ICAM-1, a finding that is supported by the enhanced clustering of LFA-1 in Cbl-b–/– T cells in response to TCR stimulation. Thus, Cbl-b plays a negative role in Crk-L-C3G-mediated Rap1 and LFA-1 activation in T cells.

Engagement of the T cell antigen receptor (TCR) 1 by the antigenic peptide plus major histocompatibility complex (MHC) in the antigen presenting cells triggers rapid tyrosine phosphorylation and activation of protein-tyrosine kinases, the Src family (Lck and Fyn), and the Syk family (Zap-70). Activation of these kinases in turn induces tyrosine phosphorylation of a number of intracellular substrates including adaptor proteins to form intermolecular network (1,2). Cbl is one of the adaptor proteins, which consists of an amino-terminal SH2-like domain, a RING finger, and carboxyl-terminal proline-rich sequences with potential tyrosine phosphorylation sites (3). Previous studies from numerous laboratories have demonstrated that Cbl associates with a number of critical signaling molecules upon T cell activation including Zap-70, Grb-2, 14-3-3, phosphatidylinositol 3-kinase (PI3K), and Crk-L (3). In addition to its adaptor's role, Cbl also functions as an E3 ubiquitin (Ub) ligase, whose RING finger domain binds to a Ub-loaded conjugation enzyme or E2, and whose other protein interaction domains recruit potential substrates; Cbl then helps transfer Ub from the E2 to the substrate (4,5). The identification of Cbl as an E3 has facilitated our understanding of Cbl in the regulation of intracellular signaling transduction.
Cbl-b was isolated as a mammalian Cbl homologue from human breast caner cells and it is ubiquitously expressed and has a very high homology with Cbl, particularly in the amino terminus and the RING finger domain (6). The importance of Cbl-b in T cell regulation is underscored by the increased proliferation and cytokine production in Cbl-b-deficient T cells (7,8). Cbl-b Ϫ/Ϫ mice also display spontaneous or antigen-induced autoimmunity. We previously showed that Cbl-b has E3 ligase activity by inducing ubiquitionation of p85, the regulatory subunit of PI3K (9). Instead of inducing degradation of p85, Ub conjugation to p85 affects its interaction with upstream molecules such as TCR chain and CD28, thus regulating the protein complex formation (10). In human Jukat T cell line, we observed that Cbl-b becomes phosphorylated on tyrosine residues and associates with Crk-L in an activationdependent manner (11). However, the physiological significance of Cbl-b and Crk-L interaction remains unclear.
Crk-L is an adaptor protein that is composed of an NH 2terminal SH2 domain and COOH-terminal two SH3 domains. The SH2 domain has been shown to bind to Cbl and/or Cbl-b in an activation-dependent manner, whereas a COOH-terminal SH3 domain constitutively associates with C3G, a guanine exchange factor, which has been shown to be a specific exchange factor for Rap-1 (12). The Cbl-Crk-L-C3G signal pathway has been proposed to play a role in Rap-1 activation and therefore in negative regulation of Ras signal pathway in T cells (13). Whether Cbl-b is also involved in Rap1 activation in T cells remains to be determined. In this study, we examined whether Cbl-b acts as an E3 ligase for Crk-L and whether Cbl-b deficiency affects Crk-L-C3G signaling in response to TCR stimulation. The study may shed light on the biological function of Cbl-b in T cell activation and tolerance induction.
Cbl-b cDNAs encoding wild-type Cbl-b, or Cbl-b RING finger mutants harboring Cys to Ala (Cbl-b CA), or Trp to Ala (Cbl-b WA) mutation, the Ub cDNA with a Myc epitope tag, and the Crk-L plasmid were described previously (9,11). The bacterial expression plasmid containing glutathione S-transferase (GST)-RalGDS Rap1 binding domain (RBD) was provided by J. Bos. Preparation of GST fusion protein was performed as described previously (4).
Mice and T Cell Activation-Cbl-b-deficient (Cbl Ϫ/Ϫ ) mice on a C57BL/6 background were originally provided by M. Naramura and H. Gu at the National Institutes of Health. Primary T cells were collected from lymph nodes of 6 -8-week-old mice. Single-cell suspensions of T cells were prepared at 3 ϫ 10 7 cells/ml in RPMI 1640 (Irvine Scientific, Irvine, CA) supplemented with 5% fetal calf serum. T cells were either left untreated or were added with 10 g/ml anti-CD3⑀. The cells were incubated on ice for 10 min before stimulation at 37°C for various times as indicated.
Cell Culture and Transfection-Jurkat-TAG T cells were grown in RPMI 1640 medium (Irvine Scientific) supplemented with 10% fetal bovine serum and antibiotics. For protein expression in Jurkat T cells, cells were transfected with an appropriate amount of plasmid (usually 1-5 g in total) by electroporation (240 V, 950 microfarads; Bio-Rad). After 48 h, cells were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM sodium pyrophosphate, 2 mM orthovanadate, and 10 g/ml each aprotinin and leupeptin) for 30 min at 4°C, and insoluble materials were removed by centrifugation at 15,000 ϫ g at 4°C for 20 min. For displaying ubiquitinated protein, 0.1% SDS and 5 mM 2-mercaptoethanol were added into lysis buffer to disrupt nonspecific protein interactions.
Adhesion Assay-96-Well Nunc Maxisorp Immuno flat-bottomed plates were coated overnight at 4°C with 10 g/ml of soluble recombinant murine ICAM-1-Fc (R&D Systems, Minneapolis, MN), washed, blocked with 2% BSA/PBS for 1 h at 37°C, and then washed with medium. 1 ϫ 10 6 purified lymph node T cells in 100 l of 2% BSA/PBS were mixed with 10 g/ml of anti-CD3⑀ or 20 ng/ml of phorbol 12myristate 13-acetate. Cells were allowed to attach for 30 min at 37°C and nonadherent cells removed with warm RPMI1640 medium. Adherent cells were quantified, and all data are expressed as the mean of the percentage of binding cells relative to total cell input from three replicate wells.
Flow Cytometric Analysis-Lymphocytes from Cbl-b ϩ/ϩ and Cbl-b Ϫ/Ϫ mice were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin-, or cychrome C-conjugated antibodies to murine CD4, CD8, and CD11a (mAb, 2D10; PharMingen). The cells were preincubated with anti-Fc␥R for 10 min and stained with the antibodies on ice for 30 min. After a final wash, cells were analyzed on a FACScan flow cytometer (BD Biosciences), and the data were analyzed by using CellQuest software.
Measurement of Soluble ICAM-1 Binding-1 ϫ 10 5 lymphocytes were treated without or with 10 g/ml anti-CD3 antibody or 10 g/ml phorbol 12-myristate 13-acetate for 30 min at 37°C. Cells were then washed in PBS containing 0.1% BSA, incubated with 10 g/ml soluble recombinant murine ICAM-1-Fc (R&D Systems) 30 min at 37°C, washed twice, and then incubated with 10 g/ml Fc-specific FITC-conjugated rabbit anti-human IgG (Jackson Immunoresearch, West Grove, PA) for 1 h at 4°C. Unbond secondary antibody was removed by washing twice and fluorescence of live cells detected using the FACScan flow cytometer.
Rap1 Activation Assay-T lymphocytes were incubated in cold RPMI 1640 with or without anti-CD3 antibody for 30 min on ice. Cells were then washed twice with cold medium and activated for the indicated time periods with 20 g/ml goat anti-hamster antibody at 37°C. 2 ϫ 10 7 cells were lysed in 500 l of ice-cold Rap1 lysis buffer containing 10% glycerol, 1% Nonidet P-40, 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate, 10 mM NaF, 10 g/ml each aprotinin and leupeptin at 4°C for 30 min. Lysates were clarified by centrifugation at 15,000 ϫ g for 20 min at 4°C. Supernatantes were incubated with 5 g of GST-RalGDS-RBD fusion protein coupled to glutathione-Sepharose beads for 2 h at 4°C. Beads were pelleted and rinsed three times with the lysis buffer, and the proteins were eluted from the beads with Laemmli sample buffer and separated by electrophoresis on a SDS-PAGE (12%), followed by transfer to polyvinylidene difluoride membranes. Affinity-purified activated Rap1 were detected by immunoblotting using anti-Rap1 antibody.
LFA-1 Clustering Assay-Purified CD4 ϩ T cells from Cbl-b ϩ/ϩ and Cbl Ϫ/Ϫ lymphocytes were incubated in cold medium with 10 g/ml anti-CD3⑀ (clone 145-2C11) on ice for 30 min. Cells were then washed twice with cold medium and cross-linked by incubating with 20 g/ml goat anti-hamster IgG antibody (Jackson Immunoresearch) at 37°C for 40 min. Cells were allowed to settle on poly-L-lysine-coated glass coverslips and fixed with 3.7% paraformaldehyde for 20 min, washed with PBS, and blocked with 2% BSA/PBS for 30 min. Samples were stained with FITC-anti-LFA-1 antibody (Southern Biotechnology Associates, Inc.) for 2 h at room temperature. LFA-1 clustering was visualized by using a confocal microscope (Bio-Rad). At least 100 T cells were counted for cap formation in each experiment.
Metabolic Labeling and Pulse-Chase Experiment-The experiment was performed by following previously published procedures (14,15). Briefly, primary lymph node T cells from wild-type and Cbl-b Ϫ/Ϫ mice were seeded at 5 ϫ 10 6 cells/well in 24-well plates in Dulbecco's modified Eagle's medium lacking methionine and cystine, plus 5% dialyzed fetal bovine serum. The cells were stimulated with anti-CD3 (5 g/ml each) and were then labeled for 1 h at 37°C by adding 100 Ci/ml The cells were then washed twice with Dulbecco's modified Eagle's medium and chased for different times in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were harvested at indicated time and the cell lysates were precleared with protein G-Sepharose for 30 min and were immunoprecipitated with anti-Crk-L or anti-IB␣. Immune complexes were resolved by SDS-PAGE, and the gels were dried and subjected to autoradiography. The radiolabeled protein bands were quantified by using an NIH Image 1.61 software.

Cbl-b Associates with Crk-L in Primary T Cells-
We previously reported that stimulation of human Jurkat T cells induces tyrosine phosphorylation of Cbl-b and its association with Crk-L (11). To examine whether similar events occur in murine primary T cells, we incubated mouse lymph node T cells with 2C11 anti-CD3 antibody alone or plus anti-CD28. Anti-CD3 stimulation indeed caused tyrosine phosphorylation of Cbl-b, as revealed by blotting anti-Cbl-b immunoprecipitates with anti-phosphotyrosine antibody (Fig. 1A, top panel). Costimulation with anti-CD28 further enhanced Cbl-b tyrosine phosphorylation. Crk-L was detected in anti-Cbl-b immunoprecipitates from anti-CD3-stimulated, or anti-CD3 plus anti-CD28-stimulated, primary T cells (Fig. 1A, middle panel). Equivalent amounts of Cbl-b were present in all the immunoprecipitaes from untreated or treated cells (Fig. 1A, bottom  panel). Similarly, when the cells were subjected to immunoprecipitation with anti-Crk-L antibody, Cbl-b was detected from samples treated with anti-CD3 antibody (Fig. 1B, top panel). The coimmunoprecipitation was further increased in samples treated with anti-CD28 costimulation. The data indicate that like in human Jurkat T cells, Cbl-b and Crk-L form a complex in murine primary T cells.
Cbl-b Promotes Crk-L Ubiquitination without Affecting Its Stability-The recent identification of Cbl family proteins as E3 ligases for its binding proteins prompted us to investigate whether Cbl-b also promotes Ub conjugation to Crk-L. To this end, we cotransfected Cbl-b, Crk-L, and Myc-Ub in Jurkat T cells. As shown in Fig. 2A resulted in the formation of high molecular smears in anti-Crk-L immunoprecipitates, when blotted with anti-Myc antibody, suggesting polyubiquitination of Crk-L. Of note, Ub conjugation to Crk-L was increased upon anti-CD3 stimulation. We previously described that mutation at the conserved Trp or Cys residues of Cbl-b RING finger domain abolishes its E3 ligase activity (9). To consist with this observation, we found that ubiquitination of Crk-L was also abrogated by coexpression with the Cbl-b WA or CA mutant (Fig. 2B). To determine a physiological role of Cbl-b in Crk-L ubiquitination, we examined whether Cbl-b deficiency affects Ub conjugation to Crk-L. Primary lymph node T cells from wild-type or Cbl-b Ϫ/Ϫ mice were either untreated or stimulated with anti-CD3 antibody. Anti-Crk-L immunoprecipitates were blotted with anti-Ub antibody. Stimulation of primary T cells induced ubiquitination of Crk-L in wild-type T cells (Fig. 2C). However, Ub conjugation to Crk-L was reduced in Cbl-b Ϫ/Ϫ T cells under the same stimulation conditions.
As previously described, ubiquitination of p85 of PI3K by Cbl-b does not cause p85 degradation (10). To examine whether Cbl-b affects the stability of Crk-L, we stimulated primary T cells from wild-type and Cbl-b Ϫ/Ϫ mice with anti-CD3 antibody for different time periods. It was found that Crk-L is a very stable protein (Fig. 3A). No apparent change in Crk-L protein level was observed even after 6-h stimulation. Cbl-b deficiency did not affect the protein level of Crk-L, as compared with wild-type T cells. The same membrane was blotted with antibodies against C3G, Zap-70, Fyn, Lck, and Grb2. There was no discernable difference in the protein levels of these proteins before or after anti-CD3 stimulation of T cells from wild-type and Cbl-b Ϫ/Ϫ mice. To further confirm that Cbl-b deficiency does not affect the protein stability of Crk-L, we performed pulse-chase experments (14) in which primary T cells were labeled first with [ 35 S]methionine, and the stability of the radiolabeled Crk-L was traced by immunoprecipitation at different time intervals after initial labeling. We found that the half-life of Crk-L was quite similar in both cell populations (Fig. 3B). As a positive control, we traced the half-life of IB␣, a well known protein that goes through proteasome-dependent degradation. IB␣ has a very short half-life, which was not affected by Cbl-b deficiency (Fig. 3B, bottom panel). The data reinforce our previous observation that Cbl-b exerts its E3 ligase activity toward its substrates without apparent proteolysis (10).
A recent study documented that stimulation of T cells with anti-CD3 or anti-CD3 plus CD28 induces self-ubiquitination and subsequent degradation of Cbl-b (16). We found, however, that stimulation of primary T cells with anti-CD3 for longer time (9 -18 h) resulted in an increase of Cbl-b protein in these cells (Fig. 3C). Costimulation with anti-CD28 did not reduce Cbl-b protein expression. The anti-CD3-induced Cbl-b expression was prominent, since blotting of the same lysates with other signaling molecules such as Cbl, Zap-70, Crk-L, or Lck did not show, or showed only slightly, changes in their protein amounts (Fig. 3C).
Enhanced Crk-L and C3G Association and Rap-1 Activation in Cbl-b Ϫ/Ϫ T Cells-Next, we investigated whether Cbl-b deficiency affects the adaptor's function of Crk-L. Crk-L associates with C3G through the SH3 domain in Crk-L and the proline-rich sequences in C3G (12). We found that this associ- ation was constitutive in primary T cells, without obvious change upon TCR stimulation (Fig. 4A, top panel). However, the interaction between Crk-L and C3G was markedly increased in Cbl-b Ϫ/Ϫ T cells, under both resting and anti-CD3 stimulated conditions. It was also reported that Crk-L associates with Zap-70 in an activation-dependent manner in Jurkat T cells (17). Similar results could be observed in primary T cells upon anti-CD3 stimulation (Fig. 4A, middle panel). This activation-dependent interaction between Crk-L and Zap-70 did not change in Cbl-b Ϫ/Ϫ T cells. This change in Crk-L-C3G association in Cbl-b Ϫ/Ϫ T cells was not due to the change in protein levels of Crk-L or C3G, since blotting of the cell lysates with respective antibodies showed equivalent amounts of Crk-L and C3G proteins (Fig. 3B).
The increased interaction between Crk-L and C3G may affect the guanine exchange factor activity of C3G. It is known that C3G is a specific guanine exchange factor for Rap1 (12). Rap1 is present in a GDP-bond form, and when activated, becomes a GTP-bound form. To examine the Rap1 activation in T cells, we employed a pull-down assay in which only the activated Rap1 can be recognized by a GST fusion protein containing RBD (18). As shown in Fig. 4C, GST-RBD specifically precipitates Rap1 from primary T cells. Stimulation of the cells with anti-CD3 increased the amounts of activated Rap1. GST-RBD precipitated more Rap1 in resting Cbl-b Ϫ/Ϫ T cells, in comparison with wild-type T cells and the activated Rap1 was further increased upon anti-CD3 stimulation in Cbl-b Ϫ/Ϫ T cells. Blotting the cell lysates with anti-Rap1 antibody showed similar amounts of Rap1. The result suggests that Cbl-b is a negative regulator for Rap1 activation.
Increased LFA-1 Activity in Cbl-b Ϫ/Ϫ T Cells-A recent study using Rap1 transgenic mice showed that Rap1 positively regulates cell adhesion through activating LFA-1 activity in T cells (19). The increased Rap1 activation in Cbl-b Ϫ/Ϫ T cells prompted us to examine whether Cbl-b deficiency affects Rap1mediated LFA-1 activation. To test this possibility, we examined the cell adhesion of primary T cells from wild-type and Cbl-b Ϫ/Ϫ mice to plate-bound ICAM-1-Fc protein. We found that T cells from Cbl-b Ϫ/Ϫ mice displayed increased attachment to ICAM-1-Fc, but not to BSA, as compared with wild-type T cells under both resting and anti-CD3 stimulated conditions (Fig. 5A). The difference in ICAM-1 binding is not due to the differential cell surface expression of LFA-1, as revealed by the equivalent amounts of anti-LFA-1 staining in T cell popula- tions from control and Cbl-b Ϫ/Ϫ mice (Fig. 5B). To further confirm that Cbl-b deficiency affects the binding ability of LFA-1 to ICAM-1, we performed FACS analysis on wild-type and Cbl-b Ϫ/Ϫ T cells using ICAM-1-Fc as a probe. The numbers of ICAM-1-Fc positive T cells were increased in both resting and anti-CD3 stimulated Cbl-b Ϫ/Ϫ T cells (Fig. 5C). Thus, Cbl-b deficiency increases LFA-1 activity, most likely through regulating Rap1 signaling.
Increased LFA-1 Clustering in Cbl-b Ϫ/Ϫ T Cells-It is established that engagement of T cells with antigen-presenting cells in the presence of antigenic peptide induces membrane clustering of TCR complex and accessory molecules (20,21). LFA-1 is one of such molecules that are recruited to the membrane microdomain, which is critical for T cell activation and adhesion (22). The increased binding of Cbl-b Ϫ/Ϫ T cells to ICAM-1 suggests that Cbl-b deficiency may also affect LFA-1 clustering. To investigate this possibility, we examined the formation of LFA-1 aggregates in purified CD4 ϩ T cells by cross-linking of the TCR with hamster anti-CD3 antibody, followed by secondary anti-hamster antibody. Although resting T cells showed uniformed staining to anti-LFA antibody around the cell surfaces, cross-linking of TCR resulted in the formation of LFA-1 clustering on T cell surface (Fig. 6A). When compared with wild-type T cells, Cbl-b Ϫ/Ϫ T cells showed more intense LFA-1 patches under anti-CD3 stimulated conditions. Under anti-CD3-stimulated condition, about 50% of the total T cells showed LFA-1 clustering in wild-type T cells (Fig. 6B). The number of LFA-1 patches was increased to 80% under the same stimulated conditions. The result suggests that Cbl-b is involved in T cell adhesion by regulating LFA-1 aggregation and activation.

DISCUSSION
In the present study, we presented data showing that Cbl-b acts as an E3 Ub ligase for Crk-L. Cbl-b promotes ubiquitination of Crk-L without affecting its stability. Rather, Cbl-b deficiency results in an increased association between Crk-L and C3G, which leads to augmented activation of Rap1, and subsequent enhancement of LFA-1 activation. The results suggest that Cbl-b plays a negative role in Crk-L-C3G signaling pathway in response to TCR stimulation.
We have previously demonstrated that Cbl-b regulates p85, the regulatory subunit of PI3K in an E3 ligase-dependent, proteolysis-independent manner (9, 10). Cbl-b-induced ubiquitination of p85 modulates its association with upstream molecules such as TCR subunits and CD28. The increased association between Crk-L and C3G in Cbl-b Ϫ/Ϫ T cells, as demonstrated in the present study, most likely results from the reduced Crk-L ubiquitination by Cbl-b deficiency, and the data further supports a notion that the Ub ligase activity of Cbl-b is uncoupled to the proteasome-dependent degradation of its substrates. At present, the molecular mechanisms by which Ub conjugation to a substrate modulates its biological function remain unclear. We propose that by helping tagging Ub to a substrate, Cbl-b may cause a structural change of its substrates, which may lead to a hindrance of their capability to recruit other binding molecules. It should be noted that the modification of Crk-L and C3G binding in Cbl-b Ϫ/Ϫ T cells is specific, since Cbl-b deficiency does not affect the association of Crk-L with Zap-70. It can be postulated that Ub conjugation to Crk-L may reside in the SH3 domains, which are required for C3G binding, not in the Zap-70-interacting SH2 domain.
It was recently reported that Cbl-b goes through self-ubiquitination and subsequent degradation, which is proposed to be a mechanism for Cbl-b-mediated T cell regulation (16). Although the present study did not address this issue directly, we found that stimulation of primary T cells with anti-CD3 did not affect the stability of Cbl-b. In contrast, anti-CD3 stimulation of primary T cells for longer period (9 -18 h) increased the protein expression of Cbl-b, but not its homologue, Cbl. In addition, further costimulation with anti-CD28 did not cause a decrease of Cbl-b protein amount in these cells. Thus, our result does not support a critical role of Cbl-b self-ubiquitination in T cell regulation. The increased protein expression of Cbl-b in T cells after longer time stimulation may instead point out a novel negative feedback mechanism, i.e. TCR engagement not only triggers Cbl-b-mediated ubiquitination pathway to help terminate the activation signal, but also increases Cbl-b protein expression to counter-balance the sustained T cell activation. This mechanism may be particularly important in the development of autoimmne diseases in which a chronic and sustained T cell activation is required for the breakdown of T cell tolerance. To support this hypothesis, it was reported that Cbl-b deficiency results in spontaneous autoimmunity or increased immune response to autoantigens (7,8).
Previous studies have documented a positive role of Cbl in Crk-L-C3G-mediated signaling (13,24). In these studies, Cbl was proposed to be an adaptor protein, which helps recruit Crk-L and C3G to form an activation complex for Rap1. In Jurkat T cells, it was shown that engagement of TCR induces Fyn activation and subsequent Cbl phosphorylation, which leads to Cbl-Crk-L association. Since Crk-L constitutively associates with C3G, it then catalyzes the conversion of the inactive GDP-bond Rap1 to the active GTP bond Rap1 (13). We have recently revisited this issue by analyzing the activation of Rap1 in Cbl Ϫ/Ϫ thymocytes. We found that Cbl deficiency augmented TCR-induced Rap1 activation, which was further supported by increased association of Crk-L with C3G and C3G membrane translocation (15). Our results indicate that Cbl is a negative regulator in Crk-L-C3G signaling. Here, we provided further evidence that Cbl-b, like Cbl, also plays a negative role in Rap1 activation in peripheral mature T cells. Thus, the data suggest a new paradigm on a functional role of Cbl proteins (Cbl/Cbl-b) in the negative regulation of Crk-L-C3G-mediated pathways.
Engagement of TCR results in rapid activation of Ras, another member of small GTPases, which is essential for T cell proliferation and cytokine production (25). One of the well established pathway tranduced by Ras is Raf/Mek/Erk cascade (25). Both Ras and Rap1 have a nearly identical effector domain. However, binding of Rap1 to Raf does not cause Raf activation. Rather Rap1 competes with Ras for Raf interaction and antagonizes Ras/Raf/Erk signaling (26). Particularly, in anergized T cells that show decreased Ras and Erk activation (27,28), the activation of Rap1 is increased (13). In addition, the activation of Fyn and Cbl-Crk-L complex formation is also increased. It is therefore proposed that Fyn-Cbl-Crk-L complex formation is responsible for Rap1 activation and T cell anergy induction (13). Although we did not address whether Cbl-b is involved in anergic signaling, our data rather suggest an opposite role for Cbl-b in Rap1 signaling.
To further contrast with a negative role of Rap1 in Ras activation in T cells, a recent study using Rap1 trangenic mice showed that Rap1 does not interfere with Raf/Erk signaling (19). In Rap1 transgenic T cells, the activation of integrins is increased, which is evidenced by enhanced cell adhesion and LFA-1 clustering (19). In addition, Rap1 has also been shown to mediate CD31-induced integrin activation (29). Inspired by these findings, we also examined T cell adhesion events and found that Cbl-b Ϫ/Ϫ T cells display a similar phenotype as Rap1 transgenic T cells. Thus, our data consist with a role of Rap1 in integrin-induced cell adhesion. It should be noted that Cbl-b also affects other signal molecules, such as Vav and PI3K, and these molecules are also involved in integrin signaling (30). Thus, the increased cell binding to ICAM-1 and LFA-1 aggregation in Cbl-b Ϫ/Ϫ T cells may reflect an integration of several signaling pathways. Obviously, further studies are needed to dissect the respective role of these pathways in Cbl-b-mediated integrin regulation.