Identification of a novel 14-3-3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha.
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 alpha(IIb)beta(3)-dependent stable adhesion and spreading. Interaction of the 14-3-3zeta adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbalpha subunit has been implicated in the control of alpha(IIb)beta(3) activation and cell spreading. In this study, we have examined potentially novel 14-3-3zeta binding sites by expressing mutant forms of GPIbalpha 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 GPIbalpha-14-3-3zeta 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 14-3-3zeta from GPIbalpha. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbalpha-14-3-3zeta binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3zeta binding site within the cytoplasmic tail of GPIbalpha that has an important functional role in regulating integrin-dependent cell spreading.
Mangin
Pierre
P
INSERM U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann, BP 36, 67065 Strasbourg Cedex, France.
David
Tovo
T
Lavaud
Vincent
V
Cranmer
Susan L
SL
Pikovski
Inna
I
Jackson
Shaun P
SP
Berndt
Michael C
MC
Cazenave
Jean-Pierre
JP
Gachet
Christian
C
Lanza
François
F
D048948:14-3-3 Proteins; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D016023:Integrins / Q000378:metabolism; D010766:Phosphorylation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism / Q000494:pharmacology
D048948:14-3-3 Proteins; D016023:Integrins; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D012694:Serine; D014446:Tyrosine 3-Monooxygenase