title: 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.
authors: Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Hardy
authors: H
authors: Schweigel
authors: Jörg
authors: J
authors: Geiger
authors: Florian
authors: F
authors: Beck
authors: Sophia
authors: S
authors: Buhs
authors: Helwe
authors: H
authors: Gerull
authors: Ulrich
authors: U
authors: Walter
authors: Albert
authors: A
authors: Sickmann
authors: Peter
authors: P
authors: Nollau
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D015539:Platelet Activation; D011499:Protein Processing, Post-Translational; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology; D058472:Receptors, Purinergic P2Y1 / Q000737:chemistry / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12 / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D018909:src Homology Domains
chemical_list: C111914:N(6)-methyl-2'-deoxyadenosine 3',5'-diphosphate; C548576:P2RY12 protein, human; D058921:Purinergic P2Y Receptor Antagonists; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D019000:Phosphotyrosine; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C117446:cangrelor; D017868:Cyclic AMP-Dependent Protein Kinases; D016285:Iloprost