Here we provide evidence that in B cells Cbl-b functions to negatively regulate BCR signaling by targeting Syk for ubiquitination. In Cbl-b–deficient B cells Syk fails to be ubiquitinated following BCR cross-linking in contrast to B cells from wild-type mice in which case BCR cross-linking leads to rapid ubiquitination of Syk. The results presented suggest that Cbl-b ubiquitinates active phosphorylated Syk and thus functions to dampen BCR signaling after signaling is initiated and thus plays a role in the normal down modulation of BCR signaling. Two additional negative regulators of BCR signaling have been described recently, namely SHIP and SHP-1. However, the targets of their regulation appear distinct from that described here for Cbl-b. SHIP, a 5′ inositol phosphatase, was first described to block BCR signaling when recruited to the low affinity Fc receptor, FcγRIIb, after the coligation of the BCR and FcγRIIb by the binding of immune complexes (40
). SHIP dephosphorylates PIP(3,4,5)P3, the phospholipid product of PI-3K activity, to PI(3,4)P2. Recently, SHIP-deficient B cells were shown to have elevated levels of PI(3,4,5)P3 (11
) increased recruitment of Btk to the plasma membrane (12
) and enhanced Ca2+
). However, ERK activation appeared unaffected in SHIP-deficient cells (13
). In contrast, Cbl-b does not appear to effect PI3K activity or PI3K-downstream effectors but does influence ERK activity. SHP-1, a tyrosine phosphatase, was recently shown to down-regulate the activation of Lyn and Lyn-induced tyrosine phosphorylation of the CD19 receptor in B cells resulting in reduced B cell activation (14
). As shown here, Cbl-b does not appear to affect Lyn activity. Thus, these three negative regulators of BCR signaling appear to target different elements of the BCR signaling pathways leading to down modulation of B cell responses. It will be of interest to understand the mechanisms which trigger the activity of these regulators and how their activities are coordinated.
Lymphocytes express both c-Cbl and Cbl-b. In T cells Cbl-b and c-Cbl appear to function at different stages of development and to target different substrates for ubiquitination. c-Cbl has been reported to target Syk for ubiquitination in human Ramos B cells upon BCR cross-linking and in this way to function as a negative regulator of B cell activation (17
). Thus, Cbl-b and c-Cbl may have somewhat redundant functions in regulating mature B cell activation through the ubiquitination of Syk. However, recent studies suggest that Cbl-b and c-Cbl may function differently and regulate different targets in developing versus mature B cells. In immature DT40 chicken B cells evidence was provided that c-Cbl negatively regulates BCR signaling through its affect on the essential adaptor protein BLNK resulting in a block in the recruitment of PLC-γ2 to BLNK and PLC-γ2 phosphorylation (41
). In contrast, Cbl-b was shown to positively regulate Btk-mediated activation of PLC-γ2 in immature DT40 B cells (24
). Evidence was also provided that Cbl-b functioned similarly in the immature mouse WEHI B cells. In control experiments, PLC-γ2 was shown to be phosphorylated to similar levels in mature Cbl-b+/+
B cells 1 min after BCR cross-linking consistent with the results presented here. Taken together these findings suggest that Cbl-b and c-Cbl may provide overlapping functions in the regulation of Syk in mature B cells but have distinct targets in immature B cells resulting in both positive and negative regulation.
The molecular mechanism by which Cbl-b facilitates the ubiquitination of Syk is not known. Ubiquitination is initiated by the linkage of ubiquitin (Ub) to an Ub-activating enzyme, E1. Ub is then transferred by E1 to an Ub conjugating enzyme, E2, that subsequently conjugates the COOH terminus of Ub to a lysine on the target protein through a isopeptide bond. The specificity of ubiquitination is largely determined by Ub protein ligases, E3s, which interact with both E2s, through RING finger domains, and the E2 substrates and thus facilitate the conjugation of Ub to the target protein (42
). Members of the Cbl family are E3 ligases that bind to the kinases targeted for ubiquitination through their TKB domains (16
). The ubiquitination of Syk by c-Cbl minimally requires the TKB and RING finger domain of c-Cbl. The TKB domain of c-Cbl binds to tyrosine 317(Y317) in the linker region between the SH2 and the catalytic domains of mouse Syk when it is phosphorylated following BCR cross-linking. A mutant form of Syk that lacks the Y317 showed an enhanced ability to interact with Igα suggesting that the association of c-Cbl with Syk blocks its ability to interact with Igα and to couple the BCR to downstream signaling pathways (43
). Based on these observations a model can be proposed for the regulation of BCR signaling by Cbl-b in which BCR cross-linking results in the phosphorylation of Igα, the recruitment of Syk to Igα through the SH2 domains of Syk and phosphorylation of Syk at multiple tyrosines including Y317. Cbl-b would bind to Syk Y317 through its TKB domain resulting in both the modulation of Syk/Ig association and the ubiquitination of Syk by an E2 conjugating enzyme bound to the RING finger domain of Cbl-b. The ubiquitinated Syk would be subsequently degraded. Consistent with this model we observe that in Cbl-b–deficient B cells Syk is not ubiquitinated and the phosphorylation of Igα and association of Igα with phosphorylated Syk is prolonged. These events correlated with the prolonged association of phosphorylated Syk with Vav, the prolonged phosphorylation of BLNK, PLC-γ2, ERK, and JNK, increased and prolonged Ca2+
fluxes, and increases in the expression of the activation marker CD69. Also consistent with a model in which Cbl-b targets Syk for degradation was the observation that the activation of Lyn was not significantly affected in Cbl-b-deficient B cells. Thus, by targeting Syk for degradation and modulating its association with Igα, Cbl-b regulates several downstream signaling pathways of Syk. It is of interest that the PI3K pathway was apparently not influenced in Cbl-b–deficient B cells. It remains to be determined if this is due to a functional redundancy of Cbl-b and c-Cbl.
Cbl-b also appeared to influence the formation of stable, signaling active BCR caps after BCR cross-linking. In T cells the polarization of the TCR after ligation and the formation of the supramolecular activation complex and the immunological synapse appear to be crucial prerequisites for T cell activation (44
). Evidence has been provided that Cbl-b influences this process by negatively regulating the coupling of the TCR to Vav-1 and downstream CDC42 and WASP, leading to actin cytoskeleton-dependent TCR clustering (45
). In B cells, the BCR has been shown to polarize after cross-linking into structures that concentrate Syk, Vav, Btk, and Rac and thus appear analogous to the T cell immunological synapses (46
). Here we show that in B cells from Cbl-b–deficient mice as compared with wild-type B cells the BCR remains in a signaling active Cap I structure for longer periods of time and less BCR is found condensed in signaling inactive Cap II structures. Although the functional significance of the Cap II structures is not known, it is possible that they represent preendocytotic structures involved in the removal of signaling inactive BCR from the surface. However, although Cap II structures often appear near intracellular vesicles that contain internalized BCR we show here that the intracellular BCR is associated with Syk while the Cap II BCR is not. Earlier studies showed that internalization of the BCR is dependent on BCR signaling although the exact nature of the signals required is not known. The studies presented here suggest that BCRs may be internalized from active Cap I structures as a part of normal down modulation of the response. As receptors are internalized and signaling dampened the BCRs remaining on the surface may condense into Cap II structures which are then internalized and degraded.
In summary, the studies presented here provide evidence that Cbl-b negatively regulates Syk through ubiquitination. Thus, Cbl-b has the potential to block BCR signaling at an early step effectively uncoupling the BCR from many downstream signaling pathways. Consequently, it will be of significant interest to determine the factors that induce and regulate Cbl-b activity.