Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins.
We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2). Further studies revealed that the change in mobility of Dok-2 was brought about by tyrosine phosphorylation. Dok-2 tyrosine phosphorylation was also found to be involved in collagen receptor, glycoprotein VI (GPVI), signaling as well as in outside-in signaling through the major platelet integrin, alpha IIIb beta 3. These studies also provided the first demonstration of posttranslational modification of 2 regulator of G protein signaling (RGS) proteins, RGS10 and 18. Phosphorylation of RGS18 was mapped to Ser49 by MS/MS analysis. This study provides a new approach for the identification of novel signaling molecules in activated platelets, providing new insights into the mechanisms of platelet activation and building the basis for the development of therapeutic agents for thrombotic diseases.
García
Angel
A
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford, OX1 3QU, United Kingdom. angel@glycob.ox.ac.uk
Prabhakar
Sripadi
S
Hughan
Sascha
S
Anderson
Tom W
TW
Brock
Chris J
CJ
Pearce
Andrew C
AC
Dwek
Raymond A
RA
Watson
Steve P
SP
Hebestreit
Holger F
HF
Zitzmann
Nicole
N
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D010446:Peptide Fragments / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D020710:RGS Proteins / Q000032:analysis / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C115089:DOK2 protein, human; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome; D020710:RGS Proteins; D044463:Receptor, PAR-1; C086309:thrombin receptor peptide SFLLRNP; D014443:Tyrosine