Functional association of PI3-kinase with platelet glycoprotein Ibalpha, the major ligand-binding subunit of the glycoprotein Ib-IX-V complex. Journal of thrombosis and haemostasis : JTH [J Thromb Haemost] Journal article | | Title | Functional association of PI3-kinase with platelet glycoprotein Ibalpha, the major ligand-binding subunit of the glycoprotein Ib-IX-V complex. | | Author(s) | Mu FT, Cranmer SL, Andrews RK, Berndt MC | | Institution | Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria Australia. | | Source | J Thromb Haemost 2009 Oct 30. | | Abstract | Summary
Background: The adhesion receptor glycoprotein (GP)Ib-IX-V, that binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes such as coagulation, inflammation, and platelet-mediated tumour metastasis. The cytoplasmic C-terminal domain of the ligand-binding GPIbalpha subunit contains binding sites for filamin (residues 561-572, critically Phe568/Trp570), 14-3-3zeta (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide (PI)3-kinase regulatory subunit, p85.
Objectives: We previously showed that, compared to wild-type receptor, deleting contiguous sequences 580-590 or 591-610, but not upstream sequences, of GPIbalpha expressed as a GPIb-IX complex in CHO cells inhibited VWF-dependent Akt phosphorylation used as a read-out for PI3-kinase activity. Pull-down experiments using GST-p85 or GST-14-3-3zeta constructs, and competitive inhibitors of 14-3-3zeta binding, suggested an independent association of 14-3-3zeta and PI3-kinase with GPIbalpha. In this study, we analyze a further panel of GPIbalpha deletion mutations within 580-610.
Results: We identify a novel deletion-mutant, Delta591-595, that uniquely disrupts 14-3-3zeta binding but retains functional p85/PI3-kinase association. Deletion of other sequences within 580-610 were less discriminatory, and either partially affected p85/PI3-kinase and 14-3-3zeta (Delta580-585, Delta586-590, Delta596-600, Delta601-605), or strongly inhibited binding of both proteins (Delta606-610).
Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14-3-3zeta in GPIb-dependent signaling or platelet functional studies involving truncation of the C-terminal residues in cell-based assays and mouse models. The Delta591-595 mutation provides another strategy for determining the function of GPIbalpha-associated 14-3-3zeta by selective disruption of 14-3-3zeta but not p85/PI3-kinase binding. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 19874472 |
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