Phosphoinositide Signaling in the Cytosol and Nucleus

项目总结。本研究旨在了解空间肌醇磷脂信号转导机制 在胞浆和胞核中。这些途径对癌症、神经发育障碍、 糖尿病，以及几种先天性疾病。在胞浆中，激动剂，如EGF(和许多其他的)，被激活 控制大多数细胞功能的信号通路。在原子核中，这些途径与已知途径是分开的 但控制影响DNA修复、细胞存活和其他事件的应激反应。 激动剂激活的PI3K信号通过组装多条通路的IQGAP1支架发生 包括PI3-激酶和ERK信号通路。然而，IQGAP如何组装特定的信号通路并不是 明白了。我们将集中于在IQGAP上组装完整的PI3K通路。这包括 PI4KIII、I型PIP5-K1、PI3K、RAS、PDK1和AKT的组装 脚手架。值得注意的是，我们发现IQGAP2和IQGAP3还组装了PI3-激酶途径组件 但结果不同。IQGAP2是癌细胞中的肿瘤抑制因子，而IQGAP1和IQGAP3促进 PI3K信号转导与细胞增殖在这里，我们将探索受体如何刺激IQGAP的组装 信号通路，重点是EGF受体和IQGAP1-PI 3-K和ERK通路。我们会 通过与微管连锁，强调微管附近的内体隔室的空间PI 3-激酶信号 微管相关蛋白4(MAP4)与IQGAP1和PI3-激酶相互作用。两者之间的联系 胞浆和胞核的PI信号是PIPKI，它在胞浆和胞核中产生PIP2 核内PI信号与细胞膜的隔膜无关。我们展示了一个核弹头 聚(A)聚合酶，星形聚合酶(用于斑点靶向PIPKI调节的聚(A)聚合酶)，与 PIPKI，并被磷脂酰肌醇-4，5-二磷酸(PIP2)激活。Star-PAP控制着约40%的基因和 受到许多信号的调节。最近，我们发现PIPKI也与肿瘤抑制因子p53结合， 而P53是PIP2的效应器。PIP2结合刺激P53‘S与其他核因子相互作用 控制P53的功能。Star-PAP和P53也受肌醇多聚磷酸多激酶(IPMK)的调节 产生与P53相关的磷脂酰肌醇-3，4，5-三磷酸(PIP3)，以及核PTEN， 使该PIP3去磷酸化。我们已经确定Star-PAP和P53是核的两个关键效应分子 肌醇磷脂在胁迫信号传递过程中的信号传递。这项建议将重点放在这一机制和影响 应激途径对Star-PAP功能的影响值得注意的是，PIPn与Star-PAP和P53密切相关 它对SDS-PAGE是稳定的，表明它是共价键的，我们将探索PIP2是如何与Star-PAP和 P53。这是共价的，还是抗变性的非常紧密的相互作用？我们的发现表明了新的途径 对于胞浆和核PI通路的潜在治疗控制，就像这些通路 在许多疾病过程中的基本含义，但重点是癌症。

PROJECT SUMMARY. This research seeks to understand spatial phosphoinositide signaling (PI) mechanisms in the cytosol and nucleus. These pathways have broad implications for cancer, neurodevelopmental disorders, diabetes, and several congenital diseases. In the cytosol, agonists, such as EGF (and many others), activate signaling pathways that control most cellular functions. In the nucleus, these pathways are separate from known membrane compartments but control stress responses that impact DNA repair, cell survival, and other events. Agonists activated PI3K signaling occurs through the IQGAP1 scaffold that assembles multiple pathways including the PI 3-kinase and Erk pathways. Yet, how the IQGAPs assemble specific signaling pathways is not understood. We will focus on the assembly of the full PI 3-kinase pathway on IQGAPs. This includes the assembly of the PI 4-kinase (PI4KIII), type I PIP 5-kinase (PIPKI), PI3K, Ras, PDK1 and Akt into the scaffold. Remarkably, we show that IQGAP2 and IQGAP3 also assemble the PI 3-kinase pathway components but with different outcomes. IQGAP2 is tumor suppressor in cancer cells whereas IQGAP1 and IQGAP3 promote PI3K signaling and cell proliferation. Here, we will explore how receptors stimulate the assembly of the IQGAPs signaling pathways with an emphasis on the EGF receptor and IQGAP1-PI 3-kinase and Erk pathways. We will emphasize spatial PI 3-kinase signaling at endosomal compartments at proximity to microtubules by linkage with microtubule associated protein 4 (MAP4) that interacts with IQGAP1 and PI 3-kinase. The link between the cytosolic and nuclear PI signaling is the PIPKI, which generates PIP2 in the cytosol and nucleus Nuclear PI signaling remarkably is not associated with membrane compartments. We showed that a nuclear poly(A) polymerase, Star-PAP (for speckle targeted PIPKI regulated-poly(A) polymerase), associates with PIPKI and is activated by phosphatidylinositol-4,5-bisphosphate (PIP2). Star-PAP controls ~40% of genes and is regulated by many signals. Recently, we have shown that PIPKI also binds to the tumor suppressor p53, and that p53 is a PIP2 effector. The binding of PIP2 stimulates p53’s interactions with other nuclear factors that control p53 function. Both Star-PAP and p53 are also regulated by inositol polyphosphate multikinase (IPMK) that generates phosphatidylinositol-3,4,5-trisphosphate (PIP3) associated with p53, and nuclear PTEN that dephosphorylates this PIP3. We have identified Star-PAP and p53 as two key effectors of nuclear phosphoinositide signaling during stress signaling. This proposal will focus on the mechanism and impact of this stress pathway on Star-PAP functions. Remarkably the PIPn is so tightly associated with Star-PAP and p53 that it is stable to SDS-PAGE suggesting a covalent linkage and we will explore how PIP2 is linked to Star-PAP and p53. Is this covalent or a very tight interaction that is resistant to denaturation? Our findings indicate new avenues for potential therapeutic control of both the cytosolic and nuclear PI pathways as these pathways have fundamental implications in many disease processes but with an emphasis on cancer.