The spleen tyrosine kinase (Syk) is essential for the activation of most blood and immune cells including platelets, and is a major player in cancer, immunological and vascular disorders [1, 2]. In platelets, Syk is activated by src family kinases (SFKs) downstream of the major platelet pattern-recognition receptors GPIbα, GPVI, and CLEC-2 . Full Syk activation requires its phosphorylation at several tyrosines (by SFKs and/or autophosphorylation) and leads to direct binding to the adaptor protein ‘Linker of Activated T cells’ (LAT) located in lipid rafts. Phosphoproteomic studies established that human Syk interacts with multiple regulatory proteins and contains ~32 phosphosites (tyrosines, serine/threonines), mostly located in the interdomain B . The phosphosites are phosphorylated upon different signals and may be important for Syk interaction with binding partners.
Recently, I showed, as part of my PhD study, that selective activation of GPIbα and GPVI by echicetin beads (EB) and convulxin, respectively, induced a Syk-dependent Ca2+ release and full platelet aggregation [5, 6]. GPIbα/GPVI- activation also increased Syk tyrosine phosphorylation on Y525/526 (Syk activation marker) and on Y352 (essential for the enhancement of Syk phosphorylation/activation). This response reached a rapid peak but then declined, indicating dephosphorylation. In these functional studies with human platelets, I demonstrated for the first time a strong, but also transient phosphorylation of Syk on S297 in response to several platelet agonists. Both S297 and Y352 are located in the interdomain B of Syk, which (based on mutational and structural information [4, 7]) is thought to be an essential allosteric regulator of Syk. Therefore, we investigated the crosstalk between platelet activating pathways and inhibitory pathways (cAMP/PKA, cGMP/PKG). Platelet activation/aggregation was, as expected, completely inhibited by established cAMP-elevating (prostacyclin analogue iloprost) and cGMP-elevating (guanylyl cyclase stimulator riociguat) agents. Very surprisingly, the initial tyrosine phosphorylation and activation of Syk was not inhibited by the PKA/PKG pathways but enhanced, indicated by an enhanced phosphorylation of Syk tyrosines 525/526 and 352 (hyperphosphorylation). In contrast, GPIbα/GPVI- induced Syk S297 phosphorylation was strongly inhibited by PKA/PKG pathways. I showed that both PKA/PKG pathways and PKC inhibition enhanced Syk Y525/526 and Y352 phosphorylation during GPVI (cvx)- and GPIbα (EB)-mediated platelet activation, whereas S297 phosphorylation was inhibited. This remarkable differential response was also validated with various functional analysis. Syk S297 phosphorylation in response to GPIbα/GPVI activation is rapid, stoichiometric, transient and significantly reduced by the PKC inhibitor GFX and by PKA/PKG, in parallel with increased Syk tyrosine phosphorylation/activity. The S297 interdomain phosphosite of Syk is targeted and reversibly regulated by important pathways such as PKA, PKG, PKC, which also affect Syk tyrosine phosphorylation and activity. While the Syk phosphotyrosine sites have been extensively studied at level of function, tyrosine kinases (SFK, Syk) and phosphatases (TULA-2), essentially nothing is known concerning the Syk S297 kinase (identified as PKC in my work) and serine/threonine (S/T) protein phosphatase. I will now investigate the S/T phosphatase responsible for Syk S297 dephosphorylation and the effect of Syk S297 phosphorylation on recruiting Syk interacting proteins in human/murine platelets.