Mu Fi-Tjen, Andrews Robert K, Arthur Jane F, Munday Adam D, Cranmer Susan L, Jackson Shaun P, Stomski Frank C, Lopez Angel F, Berndt Michael C
Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Australia.
Blood. 2008 May 1;111(9):4580-7. doi: 10.1182/blood-2007-09-111096. Epub 2008 Feb 25.
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.
血管性血友病因子（VWF）与黏附受体糖蛋白（GP）Ib-IX-V的结合介导血小板黏附于受损血管，并在心脏病发作和中风时触发血小板活化和血栓形成。GPIb-IX-V在GPIbα C末端含有不同的14-3-3ζ结合位点，涉及Ser609的磷酸化，一个上游位点涉及磷酸化的Ser587/Ser590，以及GPIbβ上一个依赖蛋白激酶A（PKA）的位点，涉及Ser166。14-3-3ζ调节GPIb-IX-V与VWF的结合亲和力，抑制14-3-3ζ的结合会阻断受体信号传导，这表明14-3-3ζ具有关键的功能作用。我们使用在中国仓鼠卵巢（CHO）细胞中表达的GPIbα缺失突变体来确定14-3-3ζ结合与另一种GPIb-IX-V相关信号蛋白磷酸肌醇3-激酶（PI3-激酶）之间的关系。涉及谷胱甘肽S-转移酶（GST）-PI3-激酶/p85亚基和GST-14-3-3ζ的下拉实验表明，这两种蛋白都与GPIbα的580至590/591至610连续序列相互作用。相对于野生型受体，删除CHO细胞中表达的GPIbα的这些序列而非上游序列，会抑制VWF/瑞斯托霉素依赖性的Akt磷酸化，证实该区域包含一个功能性的PI3-激酶结合位点。用GST-p85截短体进行的下拉实验表明，GPIbα结合区域涉及p85断裂簇区域（BCR）结构域，包含RSXSXP。然而，在GST-p85突变体构建体中，该潜在的14-3-3ζ结合序列的突变/缺失/磷酸化并未改变GPIb-IX的下拉，这表明PI3-激酶独立于14-3-3ζ与GPIbα结合；14-3-3ζ抑制剂肽R18也阻断了GST-14-3-3ζ对受体的下拉，但不影响GST-p85，并且GST-p85的下拉不受过量14-3-3ζ的影响。总之，这些数据表明GPIbα C末端通过14-3-3ζ和PI3-激酶的独立结合来调节信号传导。
