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Abstract：
Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
摘要：
磷酸肌醇3-激酶（PI3K）β（PI3Kβ）在功能上具有独特的整合来自受体酪氨酸激酶（RTK）的信号的能力，G蛋白偶联受体，和Rho家族GTPases。PI3Kβ优先考虑与各种膜束缚信号输入的相互作用的机制，然而,尚不清楚。先前的实验没有确定与膜束缚蛋白的相互作用是否主要控制PI3Kβ定位而不是直接调节脂质激酶活性。为了解决我们知识的差距，我们建立了一种检测方法,可以直接观察三种不同的蛋白质相互作用如何调节PI3Kβ,当PI3Kβ以生物学相关的构型在支持的脂质双层上呈现给激酶时.使用单分子全内反射荧光（TIRF）显微镜，我们确定了控制PI3Kβ膜定位的机制，信令输入的优先级排序，和脂质激酶激活。我们发现，在参与GβGγ或Rac1（GTP）之前，自抑制的PI3Kβ优先考虑与RTK衍生的酪氨酸磷酸化（pY）肽的相互作用。尽管pY肽强烈地将PI3Kβ定位于膜上，脂质激酶活性的刺激是适度的。在存在pY/GβGγ或pY/Rac1（GTP）的情况下，PI3Kβ活性显著增强,超出了简单增加膜定位所能解释的范围。相反，PI3Kβ通过与变构调节一致的机制被pY/GβGγ和pY/Rac1(GTP)协同激活。
