A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex.
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 14-3-3zeta-binding sites at the GPIb alpha C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)-dependent site on GPIb beta involving Ser166. 14-3-3zeta regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3zeta association blocks receptor signaling, suggesting a key functional role for 14-3-3zeta. We used deletion mutants of GPIb alpha expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3zeta 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-14-3-3zeta indicated that both proteins interacted with contiguous GPIb alpha sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb alpha 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 GPIb alpha-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 14-3-3zeta-binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb alpha independently of 14-3-3zeta; 14-3-3zeta inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3zeta but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3zeta. Together, these data suggest the GPIb alpha C-terminus regulates signaling through independent association of 14-3-3zeta and PI3-kinase.
Mu
Fi-Tjen
FT
Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Australia.
Andrews
Robert K
RK
Arthur
Jane F
JF
Munday
Adam D
AD
Cranmer
Susan L
SL
Jackson
Shaun P
SP
Stomski
Frank C
FC
Lopez
Angel F
AF
Berndt
Michael C
MC
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits; D015398:Signal Transduction
D048948:14-3-3 Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D021122:Protein Subunits; D019869:Phosphatidylinositol 3-Kinases