Reevaluation of Molecular Mechanisms of Syk Activation

Syk, a signaling node in many immune pathways, comprises tandem SH2 domains, a SH2-kinase linker and C-terminal tyrosine kinase domain. There are two models of Syk activation. The “OR-gate” model (Tsang et al., JBC 283, 32650), based on behavior of a GST-Syk fusion, contends that Syk can be fully activated by its phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model (Yan et al., Mol Cell Biol 33, 2188), based on studies of untagged ZAP70 and Syk, contends that Syk activation requires not only ppITAM binding but also phosphorylation of the SH2-kinase linker by a Src family kinase such as Lyn. We evaluated these models by assessing the ability of a ppITAM peptide and Lyn to activate unphosphorylated Syk with or without an N-terminal GST tag. The ppITAM alone induced pronounced activation of GST-Syk, but was much less effective in activating non-GST-tagged Syk. Lyn alone was capable of inducing a much higher level of activation of non-GST-tagged Syk than was the ppITAM alone. In the presence of ppITAM Lyn-mediated activation of non-GST-tagged Syk was significantly accelerated and was associated with similarly accelerated phosphorylation of Y352 in SH2-kinase linker of Syk. Although the “OR-Gate” model can account for the behavior of the GST-Syk, the behavior of non-GST-tagged Syk is consistent with the alternative model in which the conformational changes of Syk due to ppITAM binding do not fully activate Syk, but prime it for Lyn-mediated Y352 phosphorylation allowing rapid Syk activation.