Identification of a novel 14-3-3ζ binding site within the cytoplasmic tail of platelet glycoprotein Ibα

Abstract

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin ␣ IIb ␤ 3 -dependent stable adhesion and spreading. Interaction of the 14-3-3 adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIb␣ subunit has been implicated in the control of ␣ IIb ␤ 3 activation and cell spreading. In this study, we have examined potentially novel 14-3-3 binding sites by expressing mutant forms of GPIb␣ in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIb␣-14-3-3 interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3 from GPIb␣. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIb␣-14-3-3 binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3 binding site within the cytoplasmic tail of GPIb␣ that has an important functional role in regulating integrin-dependent cell spreading. IntroductionThe 14-3-3 protein family consists of ubiquitous homodimeric or heterodimeric intracellular adaptor proteins that take part in the signaling pathways of numerous biologic responses. 1 There have been 5 isoforms identified in human platelets, the ⑀ and isoforms being weakly expressed relative to the more abundant ␥, , and ␤ isoforms. 2 The crystal structure of 14-3-3 revealed the association of 2 monomers, each composed of a bundle of 9 antiparallel helices, to form a large negatively charged groove that could be implicated in interactions with other proteins. 3 The 14-3-3 proteins bind to specific phosphoserine-containing motifs, 4 and their dimeric nature allows them to act as intramolecular and intermolecular phosphorylation-dependent bridges. 5 The functions of the different isoforms in platelets are, however, still poorly understood.In a seminal study, it was reported that 14-3-3 interacted with the platelet von Willebrand factor (VWF) receptor, the glycoprotein Ib-V-IX (GPIb-V-IX) complex. 6 A binding site for 14-3-3 was found at the C-terminus of GPIb␣ within a serine-rich 606-SGHSL-610 sequence bearing similarities to phosphoserine containing 14-3-3 binding motifs. 1,7 Further work showed that the serine at position 609 was predominantly phosphorylated in resting platelets and that phosphorylation was required for 14-3-3 binding. 8 Glutathione S-transferase (GST)-14-3-3 pull-down and immunoprecipitation studies of GPIb-IX-transfected cells pointed to the existence of a separate binding or regulatory site in the GPIb␣ 570-590 ...

Improved platelet survival after cold storage by prevention of glycoprotein Ib clustering in lipid rafts

“…The cytoplasmic tail of GPIbα has multiple binding sites for 14-3-3ζ. [33][34][35] The domain of amino acids 551-564 contains the binding site for filamin A (amino acid ser559), 36 which anchors GPIbα to the membrane skeleton under resting conditions. The functional activity of 14-3-3ζ depends on its dimerization 37 and it has been indicated that a single 14-3-3ζ dimer can disrupt this interaction by competitive binding, thereby releasing GPIbα.…”

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

“…This may be compared with GST-14-3-3, where there was decreased coprecipitated 580 to 590 and much less 591 to 610 in parallel experiments ( Figure 1C), consistent with the presence of known 14-3-3-binding sites centered on phosphorylated residues at Ser587/Ser590 and Ser609 at the GPIb␣ C-terminus. 10,12,13 Together, these results indicated that both 14-3-3 and p85 interacted with contiguous GPIb␣ sequences 580 to 590 and 591 to 610.…”

“…Recent evidence suggests the signaling proteins, 14-3-3 and phosphoinositide 3-kinase (PI3-kinase), associated with the cytoplasmic domain of the receptor are intricately involved in regulating GPIb-IX-V-dependent platelet function. [6][7][8][9][10][11][12][13][14][15][16] The ultimate aim of the present work was to investigate the functional relationships between binding of 14-3-3 and that of PI3-kinase.…”

“…[6][7][8][9][10][11][12][13] One of these is at the extreme C-terminus of GPIb␣ involving phosphorylation of the penultimate residue, Ser609, and there is a second site N-terminal to this involving phosphorylation of Ser587 and Ser590. There is a further protein kinase A (PKA)-dependent binding site for 14-3-3 on GPIb␤ involving Ser166.…”

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…PKA might impact indirectly on the GPIbα/filamin interaction by phosphorylating filamin A leading to its protection against proteolysis 1. A key site in GPIbα that is phosphorylated in resting platelets and dephosphorylated during activation by an unknown kinase and phosphatase, respectively, is S609 43. GPIbα S609 phosphorylation is required for 14‐3‐3 binding and receptor signaling 44.…”