Phosphorylation at Y317 on Syk negatively regulates both ITAM- and hemITAM-mediated signaling and function in platelets

Spleen tyrosine kinase (Syk) is expressed in a variety of hematopoietic cells. Its phosphorylation regulates downstream signaling events upon stimulation of receptors containing an immunoreceptor tyrosine-based activation motif (ITAM), like glycoprotein VI, or a hemITAM, including the C-type lectin-like receptor 2 (CLEC-2). This study focuses on the role of a specific phosphorylation site, tyrosine 317, in the regulation of Syk function. Tyrosine 317 is located in the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 binds to the matching recognition sequence in the tyrosine kinase-binding domain of Cbl, an E3 ubiquitin-protein ligase. To evaluate the function of this phosphorylation site, we generated mice expressing Syk(Y317F) using the CRISPR/Cas9 technique. Platelets from homozygous Syk(Y317F) mice showed enhancement of platelet signaling and physiological responses after stimulation with collagen-related peptide (CRP) and CLEC-2 cross-linking. This enhancement did not occur after stimulation with AYPGKF, a protease-activated receptor 4 agonist, or 2-methylthioadenosine diphosphate, a purinergic agonist. CRP- or CLEC-2 monoclonal antibody-induced downstream signaling events, including phosphorylation of LAT and phospholipase C γ2, were enhanced in Syk(Y317F) platelets compared with platelets from wild-type (WT) littermates. Besides an increase in platelet responses in vitro, the time to occlusion in the FeCl3 injury model was decreased in Syk(Y317F) mice compared with WT littermates. However, there was no significant difference in the tail bleeding times. Taken together, these data reveal that tyrosine 317 negatively regulates Syk signaling and functions in mouse platelets.