Within seconds of ligation, the BCR initiates complex signaling pathways which determine cell fate (9
). One of the first demonstrable signaling events following BCR ligation is the phosphorylation of the Igα ITAM and the recruitment of Syk (25
). Genetic and biochemical evidence indicates that Syk activation is linked to several distal pathways through BLNK, which acts to assemble effectors of these pathways at the plasma membrane (21
). However, it was unknown how activated Syk coupled to BLNK. Herein, we demonstrate that BLNK is recruited by its SH2 domain directly to phospho-Y204, which lies carboxy-terminal to the Igα ITAM tyrosines. Using these chimeras, we showed that mutation of Y204 and another functionally important tyrosine, Y176, uncoupled Igα from BLNK-dependent pathways leading to PLC-γ2 and JNK activation. The activation of these pathways was reconstituted when BLNK was added back to the receptor complex. In addition, BLNK and Igα coprecipitate in a Y204-dependent manner in the endogenous BCR. Our data indicate that the close juxtaposition of kinase and substrate on the Igα cytoplasmic tail facilitates the rapid assembly and initiation of several signaling pathways.
Both Igα Y176 and Y204 contribute to coupling the BCR to PLC-γ2 and JNK activation. Mutation of either tyrosine alone resulted in a partial decrease in BLNK phosphorylation, although it did not significantly affect PLC-γ2 phosphorylation or calcium mobilization in response to maximal stimulation through the chimeric receptor. However, mutation of Y204 did attenuate calcium mobilization in response to suboptimal cross-linking (data not shown). Therefore, while both tyrosines function to mediate the phosphorylation of BLNK, Y176 cannot fully compensate for mutation of Y204. However, it is likely that there are other mechanisms that couple the BCR to BLNK activation, as demonstrated by the ability of chimeric Igα molecules which contain only the ITAM and not Y204 to mobilize intracellular calcium (14
). Our chimeric Igα receptors with a mutation of Y204 were not completely deficient at inducing BLNK phosphorylation or calcium mobilization.
Although functionally redundant, Y176 and Y204 probably link the BCR to BLNK through different mechanisms. As demonstrated, BLNK is directly recruited through its SH2 domain to phospho-Y204 following receptor ligation. This interaction was also suggested in a recent preliminary report (19
). The binding of the BLNK SH2 domain appears highly specific in that only the pYQDV motif in Igα was selected from the array of cytosolic phosphotyrosines available following receptor stimulation (Fig. and and B). Closely related motif YDDV in serine/threonine kinase HPK-1 has also been shown to bind the BLNK SH2 domain (59
). These sequences suggest a possible consensus motif of Y-hydrophilic amino acid-D-V. A BLAST search of this motif did not reveal additional potential BLNK binding partners, indicating a very limited array of potential ligands for the BLNK SH2 domain. Furthermore, similar unpaired tyrosines could not be detected within the cytosolic tails of other ITAM-containing receptors. Therefore, the non-ITAM tyrosines of Igα appear to represent a unique signaling mechanism.
In contrast to the role of phospho-Y204 in the activation of BLNK, the mechanism by which Y176 contributes to signaling is unclear. We were unable to detect any protein that specifically bound to either unphosphorylated or phosphorylated Y176. This Y176 motif (YEDE) is not part of any recognizable SH2 or PTB domain consensus sequence (4
). Furthermore, there was no detectable phosphorylation of Igα Y176 following receptor stimulation (Fig. ). Y176 is only six amino acids from Y182, the binding site for Syk, making it less likely that Y176 independently binds a distinct protein. Rather, the hydroxyl moiety of Y176 might provide a secondary binding site for BLNK or a BLNK-associated protein. This would be consistent with the relatively mild reduction in BLNK phosphorylation observed when only Y176 was mutated.
We did not determine if BLNK and Syk bound to the same or to different Igα molecules. If BLNK and Syk were bound in cis
, the amino terminus of BLNK, which contains several potential tyrosine phosphorylation sites, might be in close proximity to the catalytic domain of Syk (14
) (Fig. ). Furthermore, numerous proteins in an Igα-based complex, including Syk, BLNK, and other BLNK-associated molecules, could mediate cooperative binding events favoring the assembly of multimeric signalsomes. Alternatively, it might be difficult to accommodate both Syk and BLNK on the same Igα chain, which raises the possibility that they might be recruited to different Igα molecules. The cross-linking of the receptor forms large caps, and proteins associated with adjacent Igα molecules would be brought into close apposition, facilitating the phosphorylation of BLNK in trans
. Igβ, which has no non-ITAM tyrosines or obvious recruitment sites for BLNK, cannot compensate for mutation of Y176 and Y204 (54a
). Therefore, it is likely that only Igα recruits BLNK to the BCR.
FIG. 7. Model of Syk and BLNK cooperation in mediating PLC-γ2 and JNK activation. Upon BCR stimulation Syk is recruited to, and activated by, the phosphorylated ITAM tyrosines in the Igα cytoplasmic tail. Subsequent or concurrent recruitment of (more ...)
Both Igα and Igβ contain ITAMs, which, if phosphorylated, should recruit Syk (50
). However, this potential does not always translate into competency to signal. Igβ alone induces weak tyrosine phosphorylation (36
) and chaotic intracellular calcium mobilizations (15
). It is likely that Igβ functions to regulate Igα phosphorylation and lower the activation threshold (42
Immunoprecipitations from resting B cells demonstrate that the BCR is constitutively associated with Src family kinases and with Syk. Our observation that BLNK coimmunoprecipitates with the unstimulated BCR indicates that a proximal substrate is present as well. The preassembly of these signaling molecules at the BCR may prime the receptor for rapid activation in response to ligation. Alternatively, receptor-facilitated assembly of both kinases and substrate may be required for resting BCR function. Indeed, surface expression of membrane Ig has been demonstrated to be required for maintenance of mature B lymphocytes in the periphery (39
). The presence of BLNK may link the receptor to pathways required for survival.
In summary, our observations indicate that coupling of kinase to substrate is accomplished by the direct recruitment of BLNK to Igα. This simple and efficient solution provides an explanation for the rapidity with which the BCR can initiate signals. Furthermore, it provides a model for how receptor assembly, in the absence of ligation, could transmit signals sufficient for cell survival.