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Deciphering of ADP ‐induced, phosphotyrosine‐dependent signaling networks in human platelets by Src‐homology 2 region (SH2)‐profiling
Author(s) -
Schweigel Hardy,
Geiger Jörg,
Beck Florian,
Buhs Sophia,
Gerull Helwe,
Walter Ulrich,
Sickmann Albert,
Nollau Peter
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201200353
Subject(s) - sh2 domain , proto oncogene tyrosine protein kinase src , tyrosine phosphorylation , phosphorylation , protein tyrosine phosphatase , microbiology and biotechnology , signal transduction , receptor tyrosine kinase , platelet activation , biochemistry , biology , tyrosine kinase , tyrosine , platelet derived growth factor receptor , chemistry , platelet , receptor , immunology , growth factor
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 ( SH 2)‐profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate ( ADP ). Applying a panel of 31 SH 2‐domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P 2 Y receptors by synthetic drugs revealed a major role for the P 2 Y 1 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 P 2 Y 12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P 2 Y receptors. By SH 2 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.