Banfić H, Downes C P, Rittenhouse S E
Kimmel Cancer Institute and Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1998 May 8;273(19):11630-7. doi: 10.1074/jbc.273.19.11630.
Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin alphaIIb beta3 activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). After aggregation, a larger amount of PtdIns(3,4)P2 is generated. We report that this latter PtdIns(3,4)P2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca2+-dependent protease calpain. Elevation of cytosolic Ca2+ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KIIalpha." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the beta gamma subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to Gbeta gamma and is, in addition, activated. Both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been shown to stimulate PKBalpha/Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKBalpha/Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P2 generated after aggregation. There is thus potential for both pre- and post-aggregation-dependent signaling by PKBalpha/Akt.
血小板凝血酶受体或蛋白激酶C的刺激会引发纤维蛋白原依赖性聚集，这是整合素αIIbβ3激活的一种功能。此类血小板会迅速且短暂地形成磷脂酰肌醇3,4,5-三磷酸（PtdIns(3,4,5)P3）和少量的磷脂酰肌醇3,4-二磷酸（PtdIns(3,4)P2）。聚集后，会生成大量的PtdIns(3,4)P2。我们报告称，后一种PtdIns(3,4)P2主要通过渥曼青霉素可抑制的PtdIns3P生成，然后由PtdIns3P 4-激酶（PtdIns3P 4-K）磷酸化产生，这是一条明显依赖于Ca2+依赖性蛋白酶钙蛋白酶激活的新途径。离子载体使胞质Ca2+升高，而无整合素/配体结合，不足以激活该途径。PtdIns3P 4-K不是最近描述的“PIP5KIIα”。聚集后，磷脂酰肌醇3-激酶和PtdIns3P 4-K的细胞骨架活性增加。在聚集之前，PtdIns3P 4-K可被异三聚体GTP结合蛋白的βγ亚基负调节。聚集后，PtdIns3P 4-K钙蛋白酶依赖性地失去对Gβγ的敏感性，此外，还被激活。PtdIns(3,4,5)P3和PtdIns(3,4)P2均已被证明可通过磷酸肌醇依赖性激酶1刺激PKBα/Akt的磷酸化和激活。我们发现血小板中PKBα/Akt的激活是磷酸化依赖性的且呈双相性；初始阶段依赖于PtdIns(3,4,5)P3且更有效，而第二阶段则取决于聚集后产生的PtdIns(3,4)P2。因此，PKBα/Akt存在聚集前和聚集后依赖性信号传导的可能性。
