Regulation of Serotonin Transport in Human Platelets by Tyrosine Kinase Syk

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the regulation of numerous neuro-physiological processes. The circulating level of 5-HT is regulated by the membrane transporter SERT present both in the presynaptic nerve terminals and blood platelets. 5-HT transport is a process tightly regulated by a variety of factors including protein phosphorylation. Aim of this study was to ascertain if also the SERT Tyr-phosphorylation mediated by Syk-kinase concurs to the regulation of SERT activity. Indeed we found that 5-HT uptake decreased upon platelet exposure to piceatannol or Syk-inhibitor II, two structurally unrelated inhibitors of the tyrosine-kinase Syk. Tyr-phosphorylation of anti-SERT-immuno-stained proteins in membrane extracts and in anti-SERT-immuno-precipitates, decreased upon platelet treatment with piceatannol, in parallel with a reduction of Syk-activity. Syk was immuno-revealed in the anti-SERT immuno-precipitates, which displayed a piceatannol-sensitive kinase activity towards SERT itself and the Syk-substrate α-sinuclein. Syk inhibitors also caused a decrease of the monensin-induced 5-HT-efflux from platelets and of imipramine binding to them. It is concluded that, in addition to the phosphorylation of SERT mediated by various other kinases, also that catalyzed by Syk might play an important role in the 5-HT transport, likely favoring the transporter conformation exposing the neurotransmitter binding sites.

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…Interestingly, a reduction in imipramine binding sites was observed after PP2/SU6656 treatments, suggesting a change in SERT conformation may attend transporter Tyr phosphorylation. Pavanetto et al (2011) also reported evidence of P-Tyr incorporation in human platelet SERT by immunoblotting of SERT immunoprecipitates, with evidence presented indicating a decrease in labeling from platelets treated with the Syk kinase inhibitor piceatannol, paralleling reductions in 5-HT uptake. Corroborative findings of piceatannol-induced reductions in SERT phosphorylation were also obtained using a metabolic labeling strategy.…”

“…How these findings in rat platelets and transfected cells relate to the more rapid effects detected in human platelets is as yet unclear and should be further pursued. Rapid (minutes) inhibitory effects on 5-HT uptake have also been reported after treatment of human platelets with inhibitors of the intracellular TK Syk (Pavanetto et al, 2011), suggesting that SERT proteins, at least in platelets, may be the target of multiple cytosolic TKs. A reduced 5-HT uptake V max was reported after Syk inhibition in the latter study, which the authors attribute to stabilization of the transporter in an inward-facing conformation, due to a loss of imipramine binding sites, although the whole cell nature of the binding studies precludes elimination of SERT internalization as a possible explanation.…”

Selective serotonin reuptake inhibitors reduce P2Y12 receptor-mediated amplification of platelet aggregation

“…Syk is activated after the binding of fibrinogen to integrin α IIb β 3 [26]. Recently, Pavanetto and colleagues reported that in human platelets Syk is co-immunoprecipitated with the antibody raised against serotonin transporter and regulates the activity of serotonin transporter [27]. In addition, direct interaction between serotonin transporter and integrin α IIb β 3 has been previously identified [21].…”

Immuno-Thrombotic Effects of Platelet Serotonin

“…Consequently, Syk and 5-HTT interaction seems to be dispensable for the initial activation of GPVI complex, but enhanced Syk activity may regulate the 5-HT uptake in platelets [29].…”