Chimaerins:new receptors for DIACYGLY & phorbol esters

描述（由申请人提供）：已充分确定佛波醇酯（模拟脂质第二信使甘油二酯（DAG）作用的天然肿瘤促进剂）激活蛋白激酶C（PKC），一种在多阶段致癌作用中发挥重要作用的丝氨酸-苏氨酸激酶家族。PKC作为佛波醇酯的唯一受体的传统观点受到了与PKC无关的蛋白质的发现的挑战，这些蛋白质具有C1结构域，即PKC中的佛波醇酯结合位点。事实上，我们已经确定了“嵌合蛋白”，一个与Rac GAP（GTP酶激活蛋白）具有同源性的蛋白质家族，是佛波酯和DAG的高亲和力受体。此外，与PKC一样，β 2-嵌合蛋白同种型以PKC非依赖性方式受到佛波酯的易位。重要的是，我们发现β 2-chimaerin加速Rac 1的GTP水解，导致这种GTP酶失活。这种竞争性更新的中心假设是，通过佛波酯/DAG激活嵌合蛋白将损害Rac介导的信号传导和功能。在具体目标1中，我们将探讨DAG生成的生长因子，嵌合体易位和Rac-GTP水平之间的复杂的相互关系。我们预测，嵌合蛋白易位到质膜通过其C1结构域将导致嵌合蛋白Rac-GAP活性的激活和Rac失活。在具体目标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，以下类似的模型PKC同工酶。我们已经分离出几个嵌合蛋白相互作用的蛋白质，发挥重要作用，在控制细胞内的定位和功能的嵌合蛋白。这个目标的第二个方面将涉及在我们的实验室中分离的磷酸酪氨酸蛋白的表征，可能与β 2-嵌合蛋白SH 2结构域。在具体目标3中，我们将重点关注嵌合蛋白亚型在促有丝分裂信号传导中的作用。由于Rac在有丝分裂原性的控制中具有核心作用，我们的假设是嵌合蛋白通过抑制Rac功能，将损害有丝分裂信号传导和增殖。由于Rac对肌动蛋白细胞骨架重组至关重要，因此Specific Aim 4将探索嵌合蛋白是否调节转移级联的关键步骤，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及其与增殖、恶性转化和转移的关系描绘新的PKC非依赖性途径。更重要的是，我们的研究挑战了佛波醇酯作为细胞中选择性PKC激活剂的用途。

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.