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|Title:||A functional 14-3-3ζ-independent association of PI3-kinase with glycoprotein Ibα, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex|
|Other Titles:||A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex|
|Citation:||Blood, 2008; 111(9):4580-4587|
|Publisher:||Amer Soc Hematology|
|Fi-Tjen Mu, Robert K. Andrews, Jane F. Arthur, Adam D. Munday, Susan L. Cranmer, Shaun P. Jackson, Frank C. Stomski, Angel F. Lopez and Michael C. Berndt|
|Abstract:||Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3ζ–binding sites at the GPIbα C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3ζ regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3ζ association blocks receptor signaling, suggesting a key functional role for 14-3-3ζ. We used deletion mutants of GPIbα expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3ζ binding to another GPIb-IX-V–associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)–PI3-kinase/p85-subunit and GST–14-3-3ζ indicated that both proteins interacted with contiguous GPIb sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIbα expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase–binding site. Pull-down experiments with GST-p85 truncates indicated the GPIbα-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3ζ–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb independently of 14-3-3ζ; 14-3-3ζ inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3ζ but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3ζ. Together, these data suggest the GPIb C-terminus regulates signaling through independent association of 14-3-3ζ and PI3-kinase.|
|Keywords:||Blood Platelets; CHO Cells; Animals; Humans; Cricetulus; 14-3-3 Proteins; Platelet Glycoprotein GPIb-IX Complex; Protein Subunits; Signal Transduction; Protein Binding; Cricetinae; Phosphatidylinositol 3-Kinases|
|Description:||Copyright © 2008 by American Society of Hematology|
|Appears in Collections:||Medicine publications|
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