A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism

Abstract

Edited by Jeffrey E. Pessin Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dualphosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN protooncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAMbindingprimesSYKforrapidLYN-mediatedphosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival. SYK (spleen tyrosine kinase), 3 a cytoplasmic protein-tyrosine kinase (PTK), is crucial for mediating antigen-associated signals in various cell types of the innate and adaptive immune system (1, 2). This signal mediation is essential to the propagation and activation of hematopoietic cells such as B cells, mast cells, and platelets. Aggregation of IgE or ligand-binding receptors on the surface of the cells triggers the phosphorylation of All authors were or are employees of Merck & Co., Inc.

The Serine/Threonine Protein Phosphatase 2A (PP2A) Regulates Syk Activity in Human Platelets

“…This hypothesis is strongly supported by the crystal structures of full-length Syk (fl-Syk) as wild-type and Y348F/Y352F (YYFF) mutant forms in complex with the nonhydrolyzable ATP-analogue AMP-PNP, demonstrating the molecular nature of the autoinhibited conformation [24]. These impressive structures of fl-Syk, combined with further kinetic data [43], clearly demonstrated the existence of an autoinhibited Syk, but also suggested crucial role(s) of interdomain-B, including Y348/Y352 for the activation of Syk. Unfortunately, a considerable part (amino acids 262-337, containing other phosphorylation sites including S297) of the crucial interdomain-B was not resolved, perhaps due to the particularly flexible nature of this component [24].…”

“…Overall, this Syk interdomain-B is an important molecular switch, which could allow graduated levels of Syk activity, depending on the state of interdomain and kinase domain phosphorylation. Such graduated activity of Syk was suggested to be important for tonic signaling, where complete or long-term Syk activation may not be desirable [ 43 ]. Syk was established as a promising therapeutic target in autoimmunity and inflammation [ 52 ].…”

Fine-Tuning of Platelet Responses by Serine/Threonine Protein Kinases and Phosphatases—Just the Beginning

“…2. [54][55][56] GPVI-/GPIbα-mediated platelet activation increased the well-established Syk Y-phosphorylation/activation and the stoichiometric, transient PKC-mediated Syk S297 phosphorylation as well. PKC inhibition abolished this Syk S297 phosphorylation, but enhanced GPVI-/GPIbα-increased Syk Y-phosphorylation/activity, indicating a possible feedback inhibition.…”

“…2). [54][55][56] This sophisticated SFK-Syk cascade activates phospholipase (PLC) γ2, via Yphosphorylation, leading to elevated DAG/IP3/Ca 2þ and PKC activation. 53 Syk also stimulates ADP secretion and TxA 2 synthesis, which enhance initial platelet activation.…”

Amurensin H, a Derivative From Resveratrol, Ameliorates Lipopolysaccharide/Cigarette Smoke–Induced Airway Inflammation by Blocking the Syk/NF-κB Pathway

“…Syk is activated fully by phosphorylation or binding to ITAM via its tandem Src homology 2 (SH2) domains. Dual-phosphorylated ITAMs recruit Syk, engage Syk docks to ITAM, and undergo phosphorylation at different tyrosine residues, thus triggering kinase activation and downstream signaling (Mócsai et al, 2010; Buitrago et al, 2013; Mansueto et al, 2019). Besides, Syk also mediates signaling by novel classes of receptors that do not contain ITAM sequences.…”