Chimaerins:new receptors for DIACYGLY & phorbol esters

描述（由申请人提供）：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基甘油（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，在多阶段癌变中起重要作用。PKC作为磷酯唯一受体的传统观点受到了与PKC无关的蛋白质的挑战，这些蛋白质具有C1结构域，即PKC中的磷酯结合位点。事实上，我们已经确定了“嵌合蛋白”，一个与GTPase激活蛋白（Rac GAPs）同源的蛋白家族，是磷酯和DAG的高亲和力受体。此外，像pkc一样，β -嵌合蛋白异构体也会以pkc独立的方式受到磷酸酯的易位。重要的是，我们发现β 2-嵌合蛋白加速了Rac1对GTP的水解，导致该GTP酶失活。这种竞争性更新的中心假设是，phorbol酯/DAG激活嵌合蛋白会损害rac介导的信号传导和功能。在Specific Aim 1中，我们将探讨生长因子、嵌合蛋白易位和Rac-GTP水平产生DAG之间的复杂相互关系。我们预测嵌合蛋白通过其C1结构域转位到质膜将导致嵌合蛋白Rac- gap活性激活和Rac失活。在特异性目标2中，我们将研究蛋白质-蛋白质相互作用如何调节嵌合蛋白的功能，遵循类似于PKC同工酶的模型。我们已经分离出几个嵌合蛋白相互作用蛋白，它们在控制嵌合蛋白的细胞内定位和功能中起重要作用。这个目标的第二个方面将涉及表征磷酪氨酸蛋白分离在我们的实验室，可能与β -嵌合蛋白SH2结构域。在特异性目标3中，我们将重点关注嵌合蛋白异构体在有丝分裂信号传导中的作用。由于Rac在有丝分裂性的控制中起着核心作用，我们的假设是嵌合蛋白通过抑制Rac的功能，会损害有丝分裂的信号传导和增殖。由于Rac对肌动蛋白细胞骨架重组至关重要，因此Specific Aim 4将探讨嵌合蛋白是否调节转移级联的关键步骤，包括丝状肌动蛋白膜结构、细胞扩散、迁移和侵袭。我们的研究有可能描绘出新的不依赖pkc的phobol酯和DAG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phobol酯作为细胞中选择性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.