Unveiling ZAP-70's plan B

The finding that ZAP-70 is often expressed in the B-cell chronic lymphocytic leukemia (B-CLL) cells of patients who, in general, have a more aggressive disease course was one of the first clinically relevant data to emerge from gene-expression microarray technology.¹  Before then, the ZAP-70 protein tyrosine kinase was thought to be expressed only in T-lineage cells, associated with phosphorylated immunoreceptor tyrosine–based activation motifs (ITAMs) of the T-cell receptor ζ chain following receptor engagement.

Chen, Kipps, and their colleagues have been instrumental in furthering our understanding of the unexpected association between ZAP-70 expression and poor B-CLL prognosis. They determined that ZAP-70 enhances Ig receptor signaling in B-CLL cells.^2,3  This finding was particularly surprising because B-CLL cells express the related Syk protein whose kinase activity is 100-fold higher than that of ZAP-70,⁴  and which, unlike ZAP-70, can be activated in an Src-kinase–independent fashion.⁵ 

In this issue of Blood, Chen and colleagues shed light on this issue, demonstrating that the 2 functional SH2 domains of ZAP-70 are required for enhanced IgM signaling in B-CLL cells, whereas the ZAP-70 SH1 kinase domain is dispensable. In addressing the mechanism(s) underlying this effect, Chen and coworkers eliminate the possibility that ZAP-70 facilitates Ig-receptor signaling by competing for binding to c-Cbl, an E3 ubiquitin ligase that targets proteins for proteosomal degradation. Indeed, the enhancing role of ZAP-70 is independent of its ability to interact with c-Cbl.

The second hypothesis that the authors go on to establish is that ZAP-70, but not Syk, enhances phosphorylation of the ITAMs in the Ig signaling subunit, the CD79a/CD79b (Ig-α/Ig-β) heterodimer. They find significantly higher levels of CD79b phosphorylation following anti-μ treatment in ZAP-70–expressing B-CLL cells than in their ZAP-70–negative counterparts. This hypothesis is partially at odds with the conventional “ordered” model of TCR/BCR signaling, wherein phosphorylation of ITAM tyrosines is mediated solely by Src-family kinases. However, several lines of evidence had already suggested that, at least in T cells, this simplified model might be flawed. In murine thymocytes as well as human T cells, ZAP-70, but not Syk, was shown to promote phosphorylation of ITAMs in the TCR-ζ chain, an effect that was also independent of ZAP-70 kinase activity.^6,7 

The authors' “plan B” proposes that ZAP-70 enhances BCR signaling in B-CLL cells by promoting phosphorylation of the ITAMs in the Ig signaling subunit. In this context, it is notable that Hou et al⁸  recently showed that BCR phosphorylation and internalization are mutually exclusive events. Specifically, the same tyrosine residues in the BCR complex regulate 2 distinct fates; phosphorylation results in signaling, whereas the absence of phosphorylation results in receptor internalization.⁸  While it is crucial to determine whether this phenomenon occurs in B-CLL cells, support for this proposition comes from the recent demonstration that introduction of ZAP-70 into the ZAP-70–negative BJAB lymphoma cell line leads to decreased ligand-mediated BCR internalization, even though ectopic ZAP-70 has very low catalytic activity.⁹  Furthermore, using an analog-sensitive Syk mutant, a very recent study showed that the length of time for which anti–IgM-BCR complexes are retained at the cell surface significantly shapes signaling fates; NFκB is immediately activated, whereas activation of NFAT requires that BCR complexes and the associated Syk molecule be phosphorylated for more than 1 hour.¹⁰  These effects might be expected to be more pronounced in B-CLL cells, in which levels of CD79b are significantly lower than on normal B lymphocytes.^11,12  In this model, downstream BCR signaling in B-CLL cells is mediated by the Syk kinase, but optimal cell-surface expression and phosphorylation of the BCR requires the presence of the ZAP-70 kinase (see figure).

Many questions remain, including the phosphorylation status of the Ig-α/Ig-β heterodimer, the relative association of ZAP-70 versus Syk with the Ig-α/Ig-β heterodimer in B-CLL cells as well as in normal B cells, the ZAP-70–dependent enhancement of signaling in B-CLL cells but not normal B cells, and finally, the mechanisms responsible for the distinct role of ZAP-70 in promoting CD79b phosphorylation in B-CLL cells. Irrespective of the answers, the present work by Chen and colleagues unveils a new ZAP-70–dependent pathway wherein the catalytic activity of ZAP-70 does not appear to contribute to the aggressive nature of ZAP-70⁺ B-CLL. This has significant ramifications for the development of new therapeutic strategies for B-CLL patients, targeting ZAP-70 and/or the BCR itself.