Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3zeta.
The binding of von Willebrand factor (vWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have revealed that the signaling protein 14-3-3 zeta binds directly to the cytoplasmic domain of GP Ib alpha. In this study, the dynamic association of 14-3-3 zeta with GP Ib-IX, the phosphoinositide 3-kinase (PI 3-kinase), or both, was investigated in resting, thrombin, or vWF and botrocetin-stimulated platelets by analysis of discrete subcellular fractions. Results of this study demonstrate maximal coimmunoprecipitation of 14-3-3 zeta with GP Ib-IX in the nonstimulated cytosolic fraction and in the actin cytoskeletal fraction of thrombin- or vWF-stimulated human platelets. Immunoprecipitated 14-3-3 zeta or GP Ib from cytosolic fractions contained PI 3-kinase enzyme activity and an 85-kd polypeptide recognized by antibodies to the p85 subunit of PI 3-kinase. After platelet activation, the level of association between these species decreased in the cytosolic fraction. However, increased complex formation between 14-3-3 zeta and GP Ib-IX and between PI 3-kinase and GP Ib-IX was detected in actin cytoskeletal fractions derived from thrombin- or vWF-stimulated platelets. Recombinant glutathione S-transferase-14-3-3 zeta fusion protein (14-3-3 zeta-GST) inhibited affinity-captured PI 3-kinase enzyme activity up to 70% at 2 mcmol/L 14-3-3 zeta-GST. However, increasing concentrations up to 5 mcmol/L 14-3-3 zeta-GST resulted in the 3-fold enhancement of PI 3-kinase enzyme activity. We propose that the association between PI 3-kinase and 14-3-3 zeta with GP Ib-IX serves to promote the rapid translocation of these signaling proteins to the activated cytoskeleton, thereby regulating the formation of 3-position phosphoinositide-signaling molecules in this subcellular compartment. (Blood. 2000;96:577-584)
Munday
A D
AD
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Berndt
M C
MC
Mitchell
C A
CA
D048948:14-3-3 Proteins; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003435:Crotalid Venoms / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D015539:Platelet Activation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase; D014841:von Willebrand Factor / Q000494:pharmacology
D048948:14-3-3 Proteins; D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; C030961:botrocetin; D014446:Tyrosine 3-Monooxygenase; D019869:Phosphatidylinositol 3-Kinases; D013917:Thrombin