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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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Submitted September 6, 2007
Accepted February 19, 2008
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 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*
Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia
Puget Sound Blood Center and Division of Hematology, University of Washington, Seattle, WA, United States
Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia
Department of Human Immunology, Institute of Medical and Veterinary Science, Hanson Institute, Adelaide, SA, Australia
* Corresponding author; email: michael.berndt{at}med.monash.edu.au.
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 PKA-dependent site on GPIb involving Ser166. 14-3-3 regulates 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 employed deletion-mutants of GPIb expressed in CHO cells to define the relationship of 14-3-3 binding to another GPIb-IX-V-associated signaling protein, PI3-kinase. Pull-down experiments involving GST-PI3-kinase/p85-subunit and GST-14-3-3 indicated that both proteins interacted with contiguous GPIb sequences 580-590/591-610. 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 BCR-domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3-binding 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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