Regulation of RKIP Function

这项建议的总体目标是了解Raf Kinase抑制蛋白 (RKIP)利用磷酸化在三种功能状态之间切换，并调节细胞动态组。 RKIP是磷脂酰乙醇胺结合蛋白(PEBP)家族的典型成员。 由400多种蛋白质组成，它们在序列和结构上都进化上保守，跨越 从细菌到人类。尽管RKIP的体积相对较小(185个残基)，但它扮演着双重角色 转移癌的抑制因子和心脏功能的调节剂。重要的是，它的失调可能会导致 致病州。在该信号转导通路中，RKIP既作为感受器又作为效应器。作为效应器， RKIP根据其磷酸化状态以变构的方式调节不同的激酶的活性。 具体地说，RKIP调控哺乳动物的关键信号级联信号通路，包括MAPK和G蛋白。 偶联受体(GPCRs)。蛋白激酶C(PKC)的磷酸化将RKIP功能从 抑制Raf/MAPK信号通路抑制G蛋白偶联受体激酶2，从而上调GRK2的表达 ？肾上腺素能受体(？AR)及其下游靶蛋白蛋白激酶A(PKA)。虽然固体生物数据 但人们对其分子机制知之甚少。我们现在提出RKIP通过一本小说发挥作用 调节机制，其中RKIP的磷酸化作用于现有的盐桥并触发变构 功能切换。作为一种传感器，RKIP对MAPK和PKA信号转导的变化做出反应 通过一种未知机制的路径。已经假设RKIP将作为一个简单的 两态系统，RKIP结合到Raf(RKIPRaf)或GRK2(RKIPGRK2)。然而，令人信服的数据 我们的实验室表明，RKIP采用三种离散的功能和构象状态。额外的，高的 能量中间态(RKIPKin)是负责与激酶级联反应的中间态。基座 根据我们的数据，我们提出了一个新的正反馈环，其中RKIP靶标下游的激酶 结合并磷酸化RKIPKin状态。磷酸化RKIPKin(PRKIPKin)促进进一步的磷酸化 RKIP触发了磷酸开关。在这项提案中，我们将描述的结构和功能 RKIP处于变构状态，结合了我们在生物物理学和信号转导方面的专业知识。我们将完成 具体目标如下：1)表征磷酸化开关和RKIPGRK2状态；2)表征 变构转换到高能态的性质和功能；以及3)测试变构状态的影响 通过对癌症中RKIP功能的生物物理研究来定义。尽管眼前的目标是理解 RKIP的信号作用，在此赠款申请中开发的概念将有助于理解一部 磷酸化和变构的关系，这将是普遍重要的细胞信号和 沟通。

The overall goal of this proposal is to understand the mechanism by which Raf Kinase Inhibitory Protein (RKIP) utilizes phosphorylation to switch between three functional states and regulates the cellular kinome. RKIP is the prototypical member of the Phosphatidylethanolamine Binding Protein (PEBP) family, which comprises more than 400 proteins that are evolutionarily conserved both in sequence and structure, spanning from bacteria to humans. In spite of its relatively small size (185 residues), RKIP plays a dual role as a suppressor of metastatic cancer and as a regulator of cardiac function. Importantly, its dysregulation can lead to disease states. In the kinase signaling cascades, RKIP functions both as sensor and effector. As an effector, RKIP modulates allosterically the activity of different kinases, depending on its phosphorylation state. Specifically, RKIP regulates key mammalian signaling cascades, including MAP kinase (MAPK) and G protein- coupled receptors (GPCRs). Phosphorylation by Protein Kinase C (PKC) switches RKIP function from inhibiting Raf/MAPK signaling to inhibiting G-protein-coupled receptor kinase (GRK2), thereby up-regulating the ß-adrenergic receptor (ß-AR) and its downstream target Protein Kinase A (PKA). While solid biological data are available, little is known about the molecular mechanisms. We now propose that RKIP functions via a novel regulatory mechanism, where phosphorylation of RKIP acts on an existing salt bridge and triggers an allosteric switch of function. As a sensor, RKIP responds to changes in the MAPK and PKA signal transduction pathways through an unknown mechanism. It has been hypothesized that RKIP would function as a simple two-state system, with RKIP binding to either Raf (RKIPRaf) or GRK2 (RKIPGRK2). However, compelling data from our lab indicate that RKIP adopts three discrete functional and conformational states. The additional, high energy intermediate state (RKIPKin) is the one responsible for the interaction with the kinase cascades. Based on our data, we propose a novel positive feedback loop, where kinases that are downstream of RKIP targets bind and phosphorylate the RKIPKin state. Phosphorylated RKIPKin (pRKIPKin) promotes further phosphorylation of RKIP triggering the phospho-switch. In this proposal, we will characterize the structures and functions of RKIP in its allosteric states, combining our expertise in biophysics and signal transduction. We will accomplish the following specific Aims: 1) Characterize the phospho-switch and the RKIPGRK2 state; 2) Characterize the nature and function of the allosteric switch to a high energy state; and 3) Test the effects of allosteric states defined by biophysical studies on RKIP function in cancer. Although the immediate goal is to understand the signaling role of RKIP, the concepts developed in this grant application will help understand a novel relationship between phosphorylation and allostery that will be of general importance in cell signaling and communication.