Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

Bodnar, Richard J.; Xi, Xiaodong; Li, Zhenyu; Berndt, Michael C.; Du, Xiaoping

doi:10.1074/jbc.m208329200

Cited by 71 publications

(91 citation statements)

References 45 publications

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“…Binding of VWF to EHC Cosmc +/y and EHC Cosmc −/y platelets was analyzed as previously described (42,43). To analyze platelet activation under stimulation by different concentrations of bovine thrombin, murine platelets were washed as above.…”

Section: Methodsmentioning

confidence: 99%

Platelet biogenesis and functions require correct protein O-glycosylation

Wang¹,

Jobe

Ding³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc −/ y ). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc − /y platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbβ3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc − /y platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.

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Section: Methodsmentioning

confidence: 99%

Platelet biogenesis and functions require correct protein O-glycosylation

Wang¹,

Jobe

Ding³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…In humans and mice, lack of or dysfunction of the GP Ib-IX complex causes Bernard-Soulier syndrome (2) and abrogates thrombus formation in mouse models of thrombosis (4,5). To date, it is not entirely understood how the GP Ib-IX/vWf interaction is regulated, but this process in GP Ib-IX complex has been reported to involve posttranslational modification (6), intracelluar phosphorylation at cytoplasmic regions (7), and ectodomain shedding by membrane-associated disintegrin and metalloproteinase (ADAM) metallopeptidase (8). In addition to these molecular modifications of the complex itself, it has been realized that spatial localization of the complex in the platelet membrane mediated by the platelet glycosphingolipidenriched membranes (GEMs) plays critical roles in the GP Ib-IX/vWf interaction.…”

mentioning

confidence: 99%

Platelet Glycoprotein Ibβ/IX Mediates Glycoprotein Ibα Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear

Geng

Ran

et al. 2011

Journal of Biological Chemistry

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The localization of the platelet glycoprotein GP Ib-IX complex (GP Ib␣, GP Ib␤, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ib␤ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ib␤ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ib␣ disulfide linkage with GP Ib␤ markedly decreased the amount of the GEM-associated GP Ib␣ without altering the GEM association of GP Ib␤ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ib␣ interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ib␤/GP IX mediates the disulfide-linked GP Ib␣ localization to the GEMs, which is critical for vWf interaction at high shear.

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“…22,23 A phosphorylation-specific antibody against the C-terminal sequence of GPIb␣ (RYSGHpSL; pS indicates phosphoserine) was made by immunizing New Zealand White rabbits with a synthetic peptide based on this sequence (Mimotopes, Victoria, Australia) conjugated to keyhole limpet hemocyanin via an N-terminal Cys residue, using previously described methods. 9,[23][24][25][26] The anti-phospho-Ser609 GPIb␣ antibody was affinity-purified from serum using a combination of phosphorylated and unphosphorylated forms of the immunizing peptide, as described elsewhere. 9,[23][24][25][26] Anti-Akt and anti-phospho-Ser308 Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA).…”

Section: Antibodiesmentioning

confidence: 99%

“…9,[23][24][25][26] The anti-phospho-Ser609 GPIb␣ antibody was affinity-purified from serum using a combination of phosphorylated and unphosphorylated forms of the immunizing peptide, as described elsewhere. 9,[23][24][25][26] Anti-Akt and anti-phospho-Ser308 Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA). A mouse anti-GPIb␣ monoclonal antibody, WM23, against the extracellular domain has been reported elsewhere.…”

Section: Antibodiesmentioning

confidence: 99%

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

Andrews

Arthur

et al. 2008

Blood

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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 14-3-3ζ–binding sites at the GPIbα C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3ζ regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3ζ association blocks receptor signaling, suggesting a key functional role for 14-3-3ζ. We used deletion mutants of GPIbα expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3ζ 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–14-3-3ζ indicated that both proteins interacted with contiguous GPIbα sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIbα 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 GPIbα-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 14-3-3ζ–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIbα independently of 14-3-3ζ; 14-3-3ζ inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3ζ but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3ζ. Together, these data suggest the GPIbα C-terminus regulates signaling through independent association of 14-3-3ζ and PI3-kinase.

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Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser 166 of Glycoprotein Ibβ

Cited by 71 publications

References 45 publications

Platelet biogenesis and functions require correct protein O-glycosylation

Platelet biogenesis and functions require correct protein O-glycosylation

Platelet Glycoprotein Ibβ/IX Mediates Glycoprotein Ibα Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear

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

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