Identification of the phosphotyrosine proteome from thrombin activated platelets.
Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.
Department of Clinical Pharmacology, Royal College of Surgeon's in Ireland, Dublin, Ireland.
Patricia B
PB
Maguire
Kieran J
KJ
Wynne
Donal F
DF
Harney
Niaobh M
NM
O'Donoghue
Gillian
G
Stephens
Desmond J
DJ
Fitzgerald
D001792:Blood Platelets / Q000378:metabolism*; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional; D004792:Enzyme Precursors / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D015151:Immunoblotting; D016023:Integrins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D019000:Phosphotyrosine / Q000737:chemistry*; D015539:Platelet Activation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020543:Proteome / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000072377:Syk Kinase; D013917:Thrombin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D004792:Enzyme Precursors; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; D020543:Proteome; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; D013917:Thrombin