Feedback Regulation of Syk by Protein Kinase C in Human Platelets

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.

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

“…To investigate the possible roles of both Syk and PKC for the effect of PP2A inhibition on Syk S297 phosphorylation, we used two well-established inhibitors targeting Syk (PRT) and PKC (GFX). In contrast to our previous results with Cvx-stimulation [ 33 ], OA-induced Syk S297 phosphorylation was only minimally affected by the Syk inhibitor ( Figure 2 a) and by the PKC inhibitor ( Figure 2 b), suggesting that a protein kinase other than PKC mediates the OA-induced Syk S297 phosphorylation.…”

“…In previous studies, we observed that activation of both GPIbα or GPVI caused a strong but transient stimulation of Syk S297 phosphorylation, suggesting activity of both a very active Syk S297 protein kinase but also protein phosphatase [ 33 ]. In order to characterize this protein phosphatase further, we studied the effect of PP2A and protein phosphatase 1 (PP1) on the regulation of Syk phosphorylation using conditions recently established in our laboratory for human platelets [ 34 ].…”

“…We recently described time-dependent convulxin (Cvx), GPVI-mediated effects on Syk-activation and S297 phosphorylation [ 33 ]. Therefore, we compared the profile of Syk S297 phosphorylation induced by OA and Cvx, which differed significantly ( Figure 1 d).…”

“…Our data here demonstrate that PP2A inhibition prolongs Syk S297 phosphorylation up to 30 min after Cvx stimulation ( Figure 4 ), best seen at the time points 5–30 min after Cvx addition. The trough-like appearance of Syk S297 phosphorylation in the OA/Cvx combination ( Figure 4 aii) suggested that two components of S297 phosphorylation were present, one stimulated by the Cvx/Syk-kinase dependent PKC activation [ 33 ] and one by the alternative kinase system activated by OA. However, the OA pretreatment inhibited Syk activity ( Figure 3 , also Supplementary Figure S1 ), and may therefore partially reduce Syk S297 phosphorylation in the early phase after Cvx stimulation.…”

“…Recently, we showed that specific activation of human platelets via GPVI (convulxin) or GPIbα (echicetin beads) not only stimulated transient Syk tyrosine phosphorylation and Syk activation, but also stoichiometric, transient, PKC-mediated phosphorylation of Syk S297 [ 33 ]. Moreover, pharmacological or protein kinase A (PKA)-induced PKC inhibition abolished this Syk S297 phosphorylation, but enhanced GPVI-/GPIbα-stimulated Syk tyrosine phosphorylation/activity, suggesting that Syk S297 phosphorylation is a mechanism for Syk feedback inhibition in human platelets.…”