A novel ligand-binding site in the zeta-form 14-3-3 protein recognizing the platelet glycoprotein Ibalpha and distinct from the c-Raf-binding site.
We reported previously that the zeta-form 14-3-3 protein (14-3-3zeta) binds to a platelet adhesion receptor, glycoprotein (GP) Ib-IX, and this binding is dependent on the SGHSL sequence at the C terminus of GPIbalpha. In this study, we have identified a binding site in the helix I region of 14-3-3zeta (residues 202-231) required for binding to GPIb-IX complex and to the cytoplasmic domain of GPIbalpha. We also show that phosphorylation-dependent binding of c-Raf to 14-3-3zeta requires helix G (residues 163-187) but not helix I. Thus, the GPIbalpha-binding site is distinct from the binding sites for RSXpSXP motif-dependent ligands. Furthermore, we show that wild type 14-3-3zeta has a higher affinity for GPIb-IX complex than recombinant GPIbalpha cytoplasmic domain. Deletion of helices A and B (residues 1-32) disrupts 14-3-3zeta dimerization and decreases its affinity for GPIb-IX. Disruption of 14-3-3zeta dimerization, however, does not reduce 14-3-3zeta binding to recombinant GPIbalpha cytoplasmic domain. This suggests a dual site recognition mechanism in which a 14-3-3zeta dimer interacts with both GPIbalpha and GPIbbeta (known to contain a phosphorylation-dependent binding site), resulting in high affinity binding.
Gu
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M
Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Illinois 60612, USA.
Du
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D048948:14-3-3 Proteins; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D016296:Mutagenesis; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases