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

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 GPIbα 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 GPIbα 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 GPIbα C-terminus regulates signaling through independent association of 14-3-3ζ and PI3-kinase.

Abnormal VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from patients with von Willebrand disease type 2B

“…The platelet morphology differs, however: BSS has round giant platelets deficient in internal membranes. 33 In VWD2B, the platelets, although large, often did not appear to be spheroid. It is possible that the binding of the gain-of-function VWF in the circulation while promoting platelet elimination may also exert a negative influence during megakaryocytopoiesis, modifying the organization of intracellular DMS and reducing the extension of pseudopods so important for platelet formation.…”

“…Recent evidence from studies performed in cultures point to a role for interactions between mature MKs and stromal adhesive proteins in proplatelet formation. 25,26 Interestingly, GPIb␣ is a signaling molecule, 33 offering the possibility that the GPIb-VWF axis may play a regulatory role in MK maturation. GPIb␣ serves as an anchor between extracellular matrix proteins and the actin network; significantly, macrothrombocytopenia was improved in a BSS mouse model in which the GPIb␣ cytoplasmic domain was retained.…”

Src family kinases: at the forefront of platelet activation

“…Lipid raft localization and filamin-dependent cytoskeletal association are also required for optimal GPIb-IX-V signaling. 69,70 Src and Lyn associate with the cytoplasmic tail of GPIba and the cytoskeleton following VWF engagement ( Figure 5). 71 PI3K, FAK, SHIP-1, and PTP-1B subsequently associate with the plasma membrane in an SFK-dependent manner.…”

Current State and Novel Approaches of Antiplatelet Therapy

“…95 However, an attractive possibility exists for these ROS inhibitors, as well as nonplatelet-specific targets mentioned above, in drug-eluting stents where their actions will likely be more localized. Other intracellular binding partners for GPIb-IX-V in addition to the p85 subunit of phosphatidylinositol 3-kinase 21,22 include the regulatory protein, 14-3-3ζ, and the phosphorylation dependence, functional role, and potential for future targeting of this interaction have been previously reviewed in detail. 35 Finally, antiplatelet strategies could focus on the expression and function of platelet-specific GPVI.…”

“…[10][11][12][13][14] Signaling immediately downstream of GPIb-IX-V/GPVI involves activation of Src and Syk kinase-dependent tyrosine phosphorylation pathways and Src/Syk-dependent and Src/ Syk-independent generation of intracellular reactive oxygen species (ROS). [17][18][19][20] The cytoplasmic domains of GPIb-IX-V and GPVI directly bind to signaling or adaptor proteins, including phosphatidylinositol 3-kinase (via the p85 subunit) 21,22 and constitutively phosphorylated Lyn (via a proline-rich sequence of GPVI), 23 respectively. In the case of GPVI, ligand-induced receptor cross-linking enables active Lyn to phosphorylate Syk associated with the immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain of Fc receptor γ-chain, initiating canonical Syk-dependent signaling pathways.…”