Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets.
Growth factors and transforming proteins that activate tyrosine phosphorylation have been shown to cause an increased labeling of 3-phosphate-containing phosphatidylinositols. Turnover correlates with the formation of a complex between phosphatidylinositol 3-kinase, the activated protein-tyrosine kinase, and other proteins thought to participate in transmembrane signaling. When human platelets are treated with thrombin, labeling of 3-phosphate-containing phosphatidylinositols is stimulated with a time course and concentration dependence consistent with a role for these lipids in platelet activation. We now report that when human platelets are stimulated with thrombin, a complex forms between phosphatidylinositol 3-kinase, a protein-serine/threonine kinase, and an uncharacterized platelet membrane protein. The complex is immunoprecipitated from detergent lysates of thrombinstimulated platelets by a rabbit antiserum prepared against a peptide from the cytoplasmic domain of the mouse platelet-derived growth factor (PDGF) receptor. The antigen is not the PDGF receptor, since complex formation is not stimulated by PDGF and thrombin-induced complexes are not precipitated by another rabbit antiserum against the same peptide or by monoclonal anti-human PDGF receptor antibodies. Formation of the complex is rapid (within 30 sec) and occurs at thrombin concentrations that stimulate platelet aggregation and secretion (50% of maximal complex formation at 0.03 unit of thrombin per ml). We propose that the complex initiates formation of 3-phosphate-containing phosphatidylinositols that may function in platelet activation.
Mitchell
C A
CA
Washington University School of Medicine, Division of Hematology-Oncology, St. Louis, MO.
Jefferson
A B
AB
Bejeck
B E
BE
Brugge
J S
JS
Deuel
T F
TF
Majerus
P W
PW
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007106:Immune Sera; D066298:In Vitro Techniques; D007700:Kinetics; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols / Q000097:blood / Q000302:isolation & purification; D010770:Phosphotransferases / Q000097:blood* / Q000276:immunology; D010982:Platelet-Derived Growth Factor / Q000276:immunology; D011494:Protein Kinases / Q000097:blood; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000276:immunology; D017479:Receptors, Platelet-Derived Growth Factor; D013917:Thrombin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D010770:Phosphotransferases; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D013917:Thrombin