Our map of antigen-receptor signalling pathways is based largely on studies in which the function of signalling molecules was disrupted. This knockout approach allows the ordering of components in a pathway, but it can tell us little about feedback mechanisms that might exist. This led Rolli and colleagues at the University of Freiburg to devise an alternative approach — the reconstruction of B-cell receptor (BCR) signalling pathways in Drosophila cells. The new system has produced some surprising results, which are reported in Molecular Cell.
Interference from endogenous signalling pathways is a potential problem when expressing receptors and signalling components in another vertebrate cell type. To minimize this, Rolli et al. used Drosophila Schneider S2 cells. These cells are used commonly for large-scale protein production, so it was probable that they would be able to 'cope' with the co-transfection of several foreign genes. Expression vectors containing a copper-sulphate-inducible promoter were used to express the BCR and various signalling components in S2 cells.
According to present models, BCR signal transduction is initiated by phosphorylation of the two tyrosine residues in the ITAMs (immunoreceptor tyrosine-based activation motifs) of Igα/β (the paired signalling subunits of the BCR) by the membrane-associated SRC-family protein tyrosine kinase (PTK) LYN. This is thought to allow recruitment of the cytosolic PTK SYK and activation of downstream effector enzymes. But, by transfecting S2 cells with the BCR and various combinations of wild-type or mutated forms of Igα/β, LYN and SYK, the authors found that LYN tends to phosphorylate only the first tyrosine of the ITAM, whereas SYK can phosphorylate both tyrosines. Therefore, SYK can phosphorylate the BCR independent of LYN, and it is a more probable candidate for the first step in BCR signal transduction.
Further experiments showed that the SRC-homology 2 (SH2) domains of SYK, which bind to phosphorylated ITAMs, are required to optimize the activity of its catalytic domain. This implies that SYK can regulate its own activity, because its product — phosphorylated ITAMs — induces the further activation and recruitment of SYK. This positive-feedback loop has the potential to amplify BCR signal transduction and might have a role in the initiation of BCR signals.
With such a powerful positive-feedback mechanism in place, BCR signalling could easily get out of control. Experiments in S2 cells indicated that SHP1 (SH2-domain-containing protein tyrosine phosphatase 1) might be the crucial brake. By dephosphorylating ITAMs, SHP1 could disrupt the positive-feedback loop of SYK activation. As Michael Reth discusses in a recent review in Nature Immunology, inhibition of SHP1 by hydrogen peroxide might allow the initiation of BCR signalling.
So, the S2 'gain of function' system has provided a fresh view of early BCR signal transduction, and the authors hope that it will prove to be a useful tool for the signalling community.
ORIGINAL RESEARCH PAPER
Rolli, V. et al. Amplification of B-cell antigen-receptor signaling by a Syk/ITAM positive-feedback loop. Mol. Cell 10, 1057–1069 (2002)
Reth, M. Hydrogen peroxide as a second messenger in lymphoctye activation. Nature Immunol. 3, 1129–1134 (2002).
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Bell, J. Build your own B cell. Nat Rev Immunol 3, 6 (2003). https://doi.org/10.1038/nri992