Deciphering of ADP‐induced, phosphotyrosine‐dependent signaling networks in human platelets by Src‐homology 2 region (SH2)‐profiling

Abstract

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets.

Proteomic profiling of platelet signalling

“…A recent study by Schweigel et al used SH2-domain profiling to decipher the phosphotyrosine profile of ADP-activated platelets [9]. Using 31 SH2-domains, the authors describe how the concurrent activation of protein kinase A (PKA) by the P2Y receptors abolished ADP-induced tyrosine phosphorylation, summarizing that PKA activity is negatively regulated by P2Y12, and that the P2Y receptors act synergistically to govern tyrosine phosphorylation [9].…”

“…Dok-1 GPVI, αIIbβ3 IP, 1-DE, 2-DE, LC-MS/MS [34,62] SPIN90 GPVI IP, 1-DE, 2-DE [34] Osteoclast-stimulating factor 1 GPVI IP, 1-DE, 2-DE [34] b-Pix GPVI IP, 1-DE, 2-DE [34] Grb2 GPVI IP, 1-DE, 2-DE [34,67] Teneurin-1 GPVI Ultracentrifugation, LC-MS/MS [35] Van Gogh-like protein 1 GPVI Ultracentrifugation, LC-MS/MS [35] Cortactin GPVI 2-D DIGE, MALDI, LC-MS/MS [31] Aldose reductase GPVI 2-D DIGE, MALDI, LC-MS/MS [31] ERp57 GPVI 2-D DIGE, MALDI, LC-MS/MS [31] Dok-2 CLEC-2, PAR-1 MAPK kinase kinase PAR-1 and -4, TLR2 1-DE, 2-DE, MALDI [46] HIP-55 PAR-1 and -4, TLR2 IEF/1-DE [50] RGS10 PAR-1 2-DE, LC-MS/MS [48] RGS18 PAR-1 2-DE, LC-MS/MS [48] PECAM-1 P2Y SH2-domain profiling [9] CD84 P2Y SH2-domain profiling [9] G6b P2Y SH2-domain profiling [9] DAPP1 P2Y SH2-domain profiling [9] TGF1I1 P2Y SH2-domain profiling [9] CASS4 P2Y SH2-domain profiling [9] PTPN18 P2Y SH2-domain profiling [9] ADT2 P2Y SH2-domain profiling [9] PDPK-1 αIIbβ3 1-DE, LC-MS/MS, IMAC [59] G6f αIIbβ3 1-DE, LC-MS/MS [62] G6b-B αIIbβ3 1-DE, LC-MS/MS [62] Dok Review following antibody addition, including the Src substrate cortactin and aldose reductase (AR). Cortactin is a scaffold protein phosphorylated by Src kinase [39] and the inhibition of AR results in reduced platelet aggregation.…”

“…The Src homology 2 (SH2) domain is the most prevalent of these modules, playing a central role in tyrosine kinase signaling pathways [9,27]. The development of quantitative, highthroughput phosphotyrosine SH2 profiling through the generation of GST-SH2 fusions is an alternative/complementary strategy for comprehensive profiling of the tyrosine phosphoproteome [28].…”

“…ADP binds to platelets via the purinergic G proteincoupled receptors (GPCRs) P2Y1 and P2Y12. The activation of both of these receptors is necessary for full platelet aggregation [9].…”

State of the Art in Platelet Function Testing

“…To identify platelet surface proteins, these can be enriched using lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography and free flow electrophoresis prior to identification [187]. Proteome analysis is useful in deciphering signaling cascades in human platelets [188,189]. Combination of techniques to identify posttranslational modifications with techniques of proteomics led to the identification of palmitoylated platelet proteins and platelet phospho-proteom [190].…”