Molecular Mechanism of the Syk Activation Switch

Abstract

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340 -356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.Syk is a tyrosine kinase that functions immediately downstream of antigen receptors in immune cells including B lymphocytes, mast cells, and macrophages (1-3). The central role of Syk in cell types associated with disorders such as rheumatoid arthritis and allergic rhinitis suggests that strategies to block Syk activation may have therapeutic benefit (4 -6). After receptor ligation and phosphorylation, Syk becomes localized to immune receptors and proceeds to phosphorylate downstream targets leading to Ca 2ϩ mobilization, initiation of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades, and activation of transcription factors such as NF-B (7). Although increased Syk activity is known to be indispensable for each of these cellular events, a molecular-level understanding of the steps leading to Syk activation has not been clearly defined.The Syk domain structure consists of an N-terminal pair of Src homology 2 (SH2) 2 domains separated by an inter-SH2 linker, an SH2-domain-kinase linker, and a C-terminal kinase domain. Recruitment of Syk to immune receptors involves binding of the tandem SH2 domains of Syk to motifs in the receptor known as immune tyrosine activation motifs (ITAMs), which are two YXXL sequences typically separated by 7-12 intervening residues (8, 9). Syk is known to have multiple ...

Nonredundant Roles of Src-Family Kinases and Syk in the Initiation of B-Cell Antigen Receptor Signaling

“…The observed independence of Syk phosphorylation at Y346 from the catalytic activity of Syk also suggests that Syk recruitment to the proximity of the SFKs at the BCR is not regulated by Syk catalytic activity. The finding that Syk phosphorylated ITAMs more efficiently than ZAP70 in some assays (14,39) can be explained by the higher intrinsic catalytic activity of Syk (67) and/or higher affinity of its SH2 domains for phosphorylated ITAMs (68), not necessarily by the specificity of Syk for the ITAMs.…”

“…The recruitment of these kinases to the plasma membrane is mediated by the binding of their tandem SH2 domains to phosphorylated ITAMs in the immunoreceptor-associated chains (11)(12)(13). Activation of Syk is achieved via binding to phosphorylated ITAM and/or by phosphorylation of tyrosine residues in the interdomain B between the C-terminal SH2 domain and the kinase domain (e.g., Y346 for murine Syk) (14)(15)(16). These tyrosines seem to be phosphorylated mainly by SFKs, although autophosphorylation of these sites has also been observed under various conditions (14,17,18).…”

“…Activation of Syk is achieved via binding to phosphorylated ITAM and/or by phosphorylation of tyrosine residues in the interdomain B between the C-terminal SH2 domain and the kinase domain (e.g., Y346 for murine Syk) (14)(15)(16). These tyrosines seem to be phosphorylated mainly by SFKs, although autophosphorylation of these sites has also been observed under various conditions (14,17,18). Similar to SFKs, Syk family kinases contain (auto)phosphorylation sites in the activation loop (Y519/ Y520 for murine Syk) (17).…”

“…Indeed, Syk was reported to phosphorylate both ITAM tyrosines in Iga when coexpressed in the insect cell line S2 (15), and Syk, but not its T cell paralogue ZAP70, was reported to phosphorylate ITAMs in vitro and in COS cells (14,39). Moreover, ectopic expression of Syk was able to rescue TCR signaling in the J.CaM1.6 T cell line, which is deficient in SFK Lck (40).…”

Biophysical and Mechanistic Insights into Novel Allosteric Inhibitor of Spleen Tyrosine Kinase

“…Spleen tyrosine kinase (Syk), a non-receptor-tyrosine kinase, binds to the phosphorylated ITAMs and is subsequently phosphorylated by LynB. ITAM binding (ITAM-Syk) or phosphorylation (P-Syk) results in Syk kinase activation (1,2) and the subsequent phosphorylation of direct (PI3K, PLC␥, VAV, and SLP76) and indirect (AKT, B-cell linker protein (BLNK), PKC␣ and PYK2 among others) downstream targets (3)(4)(5). Syk activation commits B cells and mast cells to an immune response (6,7) and is, therefore, an important control point for therapeutic intervention of diseases arising from inflammation, such as the common allergic response, or autoimmune responses, such as rheumatoid arthritis and asthma.…”