Chimaerins: new receptors for diacylglycerol & phorbol esters

描述(申请人提供)：众所周知，佛波酯是一种天然的肿瘤促进剂，可以模拟脂质第二信使二酰甘油(DAG)的作用，激活蛋白激酶C(PKC)，这是一种丝氨酸-苏氨酸激酶家族，在多阶段癌症发生中发挥重要作用。传统的认为PKC是佛波酯的唯一受体的观点受到了挑战，因为发现了与PKC无关的蛋白质，这些蛋白质含有一个C1结构域，即PKC中的佛波酯结合部位。事实上，我们已经证实，与RAC GAP(GTP酶激活蛋白)同源的一族蛋白质“嵌合体”是佛波酯和DAG的高亲和力受体。此外，像PKC一样，β2-嵌合体异构体可以通过佛波酯以不依赖PKC的方式进行易位。重要的是，我们发现Beta2-嵌合体加速了rac1的GTP水解，导致了这个GTP酶的失活。这种竞争性更新的中心假设是，佛波酯/DAG激活嵌合体将损害RAC介导的信号和功能。在具体目标1中，我们将探索生长因子产生DAG、嵌合体易位和RAC-GTP水平之间的复杂相互关系。我们预测嵌合素通过其C1区移位到质膜上将导致嵌合素RAC-GAP活性的激活和RAC的失活。在特定的目标2中，我们将按照与PKC同工酶描述的模型类似的模型，研究蛋白质-蛋白质相互作用如何调节嵌合体功能。我们已经分离了几个嵌合体相互作用蛋白，它们在控制嵌合体的细胞内定位和功能方面发挥着重要作用。这个目标的第二个方面将涉及我们实验室分离的可能与Beta2-嵌合体SH2结构域相关的磷酸酪氨酸蛋白的特征。在具体目标3中，我们将重点介绍嵌合体异构体在有丝分裂信号中的作用。由于RAC在有丝分裂的控制中起着核心作用，我们的假设是嵌合体通过抑制RAC功能，将损害有丝分裂信号和增殖。由于Rac对肌动蛋白细胞骨架的重组至关重要，因此特定目标4将探索嵌合体是否调控转移级联反应的关键步骤，包括微丝肌动蛋白膜结构、细胞扩散、迁移和侵袭。我们的研究有可能为佛波醇酯和DAG描绘新的PKC非依赖通路，以及它们与增殖、恶性转化和转移的关系。更重要的是，我们的研究挑战了佛波酯在细胞中作为选择性PKC激活剂的使用。

DESCRIPTION (provided by applicant): It is well established that the phorbol esters, natural tumor promoters that mimic the actions of the lipid second messenger diacylglycerol (DAG), activate protein kinase C (PKC), a family of serine-threonine kinases that play important roles in multistage carcinogenesis. The traditional view of PKC as the sole receptor for the phorbol esters has been challenged by the discovery of proteins unrelated to PKC, which have a C1 domain, the phorbol ester binding site in PKC. Indeed, we have established that "chimaerins", a family of proteins with homology to Rac GAPs (GTPase Activating Proteins) are high affinity receptors for phorbol esters and DAG. Moreover, like PKCs, the beta2-chimaerin isoform is subject to translocation by phorbol esters in a PKC-independent fashion. Importantly, we found that beta2-chimaerin accelerates GTP hydrolysis from Rac1, leading to the inactivation of this GTPase. The central hypothesis of this competing renewal is that chimaerin activation by phorbol ester/DAG will impair Rac-mediated signaling and functions. In Specific Aim 1 we will explore the complex interrelationship between DAG generation by growth factors, chimaerin translocation and Rac-GTP levels. We predict that translocation of chimaerins to the plasma membrane through its C1 domain will result in the activation of chimaerin Rac-GAP activity and Rac inactivation. In Specific Aim 2 we will investigate how protein-protein interactions regulate chimaerin function, following analogous models to those described for PKC isozymes. We have isolated several chimaerin interacting proteins that play important roles in controlling intracellular localization and function of chimaerins. A second aspect of this aim will involve the characterization of phosphotyrosine proteins isolated in our laboratory that may associate to the beta2-chimaerin SH2 domain. In Specific Aim 3 we will focus on the roles of chimaerin isoforms in mitogenic signaling. Since Rac has a central role in the control of mitogenicity, our hypothesis is that chimaerins, by inhibiting Rac function, will impair mitogenic signaling and proliferation. Since Rac is critical for actin cytoskeletal reorganization, Specific Aim 4 will explore if chimaerins regulate key steps of the metastatic cascade, including filament actin membrane structures, cell spreading, migration and invasion. Our research has the potential for delineating novel PKC-independent pathways for the phorbol esters and DAG and their relationship to proliferation, malignant transformation, and metastasis. More importantly, our research challenges the use of phorbol esters as selective PKC activators in cells.