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Blood, 1 May 2008, Vol. 111, No. 9, pp. 4580-4587.
Prepublished online as a Blood First Edition Paper on February 25, 2008; DOI 10.1182/blood-2007-09-111096.
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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
A functional 14-3-3 –independent association of PI3-kinase with glycoprotein Ib , the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex
Fi-Tjen Mu1,
Robert K. Andrews1,
Jane F. Arthur1,
Adam D. Munday2,
Susan L. Cranmer3,
Shaun P. Jackson3,
Frank C. Stomski4,
Angel F. Lopez4, and
Michael C. Berndt1
1 Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Australia;
2 Puget Sound Blood Center and Division of Hematology, University of Washington, Seattle;
3 Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Australia; and
4 Department of Human Immunology, Institute of Medical and Veterinary Science, Hanson Institute, Adelaide, Australia
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 14-3-3 –binding sites at the GPIb 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. 14-3-3 regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3 association blocks receptor signaling, suggesting a key functional role for 14-3-3. We used deletion mutants of GPIb expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3 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–14-3-3 indicated that both proteins interacted with contiguous GPIb sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb 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 GPIb-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 14-3-3–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb independently of 14-3-3; 14-3-3 inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3 but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3. Together, these data suggest the GPIb C-terminus regulates signaling through independent association of 14-3-3 and PI3-kinase.
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