Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3ζ

Adam D Munday, Michael C Berndt, Christina Anne Mitchell

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90 Citations (Scopus)

Abstract

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 14-3-3ζ binds directly to the cytoplasmic domain of GP Ibα. In this study, the dynamic association of 14-3-3ζ 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 143-3ζ with GP Ib-IX in the nonstimulated cytosolic fraction and in the actin cytoskeletal fraction of thrombin- or vWF-stimulated human platelets. Immunoprecipitated 14-3-3ζ 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 14-3-3ζ 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-14-3-3ζ fusion protein (14-3-3ζ-GST) inhibited affinity- captured PI 3-kinase enzyme activity up to 70% at 2 μmol/L 14-3-3ζ-GST. However, increasing concentrations up to 5 μmol/L 14-3-3ζ-GST resulted in the 3-fold enhancement of PI 3-kinase enzyme activity. We propose that the association between PI 3-kinase and 14-3-3ζ 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. (C) 2000 by The American Society of Hematology.
Original languageEnglish
Pages (from-to)577-584
Number of pages8
JournalBlood
Volume96
Issue number2
Publication statusPublished - 2000

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