Human signaling protein 14-3-3ζ interacts with platelet glycoprotein Ib subunits Ibα and Ibβ

David Calverley, Terrance J. Kavanagh, Gerald J. Roth

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Abstract

The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor and its arterial subendothelial von Willebrand factor (vWF) ligand. The intracellular signaling immediately following GPIb receptor occupancy connecting the adhesive event to platelet activation and aggregation has not been well characterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous protein family with diverse biologic roles, including functional modulation of several prominent signaling proteins. We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3ζ and provide evidence for the potential signaling role of this protein. Two-hybrid interactions suggest that platelet 14-3-3ζ associates with the cytoplasmic domain of GPIb subunits Ibα and Ibβ in transformed yeast cells. The 14-3-3 interaction with GPIbβ may be partly mediated through the latter's phosphorylated serine 166 residue as its mutagenesis results in 20% to 40% reduced interaction. There was 51% to 59% reduced interaction between GPIb and three 14-3-3ζ deletion mutants compared with full-length 14-3-3ζ, suggesting that either the terminal dimerization or membrane-binding domains or more than one noncontiguous 14-3-3ζ element may be required for optimal GPIb interaction. Confocal studies of platelets and a megakaryocyte cell line provided additional evidence for interaction of 14-3-3ζ with GPIbα and GPIbβ. We also found that, similar to the signaling mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3- 3ζ content is increased following vWF and ristocetin stimulation. We suggest that platelet 14-3-3ζ interacts with GPIbα and Ibβ that this interaction may be partly mediated through phosphoserine recognition, and that 14-3-3ζ cytoskeletal translocation may serve as a GPIb post-receptor occupancy signaling event.

Original languageEnglish (US)
Pages (from-to)1295-1303
Number of pages9
JournalBlood
Volume91
Issue number4
Publication statusPublished - Feb 15 1998
Externally publishedYes

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ASJC Scopus subject areas

  • Hematology

Cite this

Calverley, D., Kavanagh, T. J., & Roth, G. J. (1998). Human signaling protein 14-3-3ζ interacts with platelet glycoprotein Ib subunits Ibα and Ibβ. Blood, 91(4), 1295-1303.