Dissecting the Divergent Functions and Dynamics of ZAP-70 and SYK

Abstract

Activation of signaling pathways in immune cells can be initiated by binding of membrane proteins, known as B-cell receptors (BCRs) or T-cell receptors (TCRs), to fragments of an antigen. The proteins spleen tyrosine kinase (Syk) and zeta-chain associated protein kinase 70 (ZAP-70) are among the first proteins to be activated upon engagement of the BCR or TCR.Recruitment of these proteins to phosphorylated immunoreceptor tyrosine activation motifs (ITAMs) associated with the TCR or BCR activates the kinase whereupon it initiatesdownstream signaling pathways, eventually leading to an immune response. Syk and ZAP-70 are paralogs, with Syk being present in B-cells and ZAP-70 in T-cells. Additionally, ZAP-70 and Syk share the domain architecture of tandem SH2 domains (tSH2) linked to a kinase domain. The tSH2 is responsible for binding the phosphorylated ITAMs. Despite similarities in the structure and function of these two kinases, there are key differences in their intrinsic activity and specificity. Using the technique of hydrogen exchange by mass spectrometry(HX/MS) we have identified the Inter-SH2 linker as being significantly different in ZAP-70 compared to Syk. HX/MS experiments indicate that the Inter-SH2 linker of the Syk tSH2 is very flexible and potentially disordered unless bound to a phosphorylated ITAM. This is not the case for the ZAP-70 tSH2, which shows protection even in the apo state. These findings suggest that the intrinsic flexibility of these proteins may be the cause of some of the observed differences in auto-inhibition, activity, and specificity.