Abstract
The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.
Highlights
Human platelets are small circulating blood cells, which control, monitor, and preserve the integrity of the vessel wall
Our previous phosphoproteomic studies with human platelets showed that adenosine diphosphate (ADP) induced spleen tyrosine kinase (Syk) serine phosphorylation at S297, which is located in the interdomain-B of Syk [26]
Using a phosphospecific antibody against this site, we investigated the regulation of this phosphorylation site by ADP, by its functional inhibitor and by agonists, which activate platelets via immunoreceptor tyrosine-based activation motif (ITAM)-/Syk-dependent mechanisms
Summary
Human platelets are small circulating blood cells, which control, monitor, and preserve the integrity of the vessel wall. Distinct platelet receptors sense and bind newly exposed proteins immobilized within the extracellular matrix or on activated endothelial cells This is followed by receptor-mediated platelet activation resulting in a plethora of cellular responses, including cytoskeletal remodeling, integrin activation (e.g., integrin αIIbβ3), degranulation, synthesis/release of thromboxane A2 (TxA2), and surface exposure of anionic phospholipids, leading to platelet shape change, adhesiveness, aggregation, and coagulant activity to form a protective thrombus [2,3]. These essential physiological processes are often impaired in diseases and are tightly controlled by numerous hormones, vasoactive factors, and adhesive proteins, which activate, modulate, or inhibit these platelet functions. GPCR-coupled agonists activate phospholipase (PLC) β, elevate cytosolic Ca2+ concentration, and activate Ca2+-dependent protein kinases, such as protein kinase C (PKC), thereby inducing platelet activation [4,5]
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