PDF(Pubmed)
Abstract：
Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.
摘要：
细胞途径控制趋化性，增长,生存,和肿瘤发生被Ras超家族的受体酪氨酸激酶和小G蛋白激活，这些蛋白刺激磷脂酰肌醇3激酶（PI3K）的特定同工型。这些PI3K脂质激酶磷酸化组成性脂质磷脂酰肌醇-4,5-二磷酸（PIP2）以产生信号脂质磷脂酰肌醇-3,4,5-三磷酸（PIP3）。在直接理解方面取得了进展，受体对PI3K的适度激活。相比之下,受体和Ras协同激活PI3K至更高水平的机制尚不清楚，并提出了两种竞争模型：膜募集与膜结合酶的激活。为了解决这个中心机制问题，这项研究采用单分子成像技术来研究在支持的脂质双层上重建的六组分途径中的PI3K激活.研究结果表明，通过受体激活环（来自血小板衍生的生长因子受体，受体酪氨酸激酶）和H-Ras产生强烈的，协同激活PI3Kα，通过膜募集机制产生净激酶活性的大幅增加。协同作用需要受体磷酸-Tyr和两种阴离子脂质（磷脂酰丝氨酸和PIP2）才能使PI3Kα胜任双层对接，以及随后结合和磷酸化底物PIP2以产生产物PIP3。协同作用还需要募集到膜结合的H-Ras，这大大加快了稳定的形成，膜结合PI3Kα复合物，适度放缓其关闭速度，并显著增加其平衡表面密度。令人惊讶的是,H-Ras结合显著抑制膜结合PI3Kα分子的特异性激酶活性,但是这种微小的酶抑制作用被膜募集的显着增强所淹没。这些发现对趋化性领域有直接影响，先天免疫,炎症,致癌作用，和药物设计。
