Regulation of apoptosis by mutationally activated B-Raf

描述（由申请人提供）：突变激活的BRAF在多种人类癌症中表达，在黑色素瘤中具有惊人的高患病率。 突变的BRAF促进RAF->MEK->ERK MAP激酶信号传导途径的持续活化，这反过来促成黑素瘤细胞的许多异常行为。 使用BRAF的条件活性形式，已经证明BRAF->MEK->ERK信号传导可以抑制多种哺乳动物细胞中的细胞凋亡。 这些数据与显示多种凋亡缺陷的5/WF缺失合子小鼠的胚胎致死表型一致。 BCL-2家族蛋白，包括促凋亡和抗凋亡成员，在细胞凋亡的调节中发挥重要作用。 因此，我们发现，我们已经鉴定了BRAF的活化和仅BH 3结构域蛋白BIM的磷酸化之间的直接生物化学联系，该蛋白BIM是BCL-2家族的促凋亡成员，在黑素细胞凋亡中起重要作用。 在这里，我们提出的实验来测试的假设，BRAF激活可以影响正常黑素细胞和黑色素瘤细胞通过BIM磷酸化的改变凋亡。 为了验证这一假设，我们将利用黑素细胞中BRAF信号传导的条件激活系统。 该系统将用于探索BRAF激活在黑素细胞中的整体效应以及BRAF对细胞凋亡的特异性效应。 为了补充这些研究，我们将使用BRAF->MEK->ERK信号传导或BIM表达的遗传（RNAi）和药理学抑制剂来评估该途径在响应营养因子剥夺、基质分离或DNA损伤的黑素细胞/黑素瘤细胞凋亡中的重要性。 虽然有强有力的遗传和生物化学证据表明BRAF信号在人类黑色素瘤的发生和发展中起重要作用，BIM在黑色素细胞存活的调节中起重要作用，但我们将测试这些蛋白质在直接生物化学途径中相关联的假设。 我们将阐明BRAF调节哺乳动物细胞中BIM促凋亡作用的生化机制，重点是磷酸化在调节BIM表达/活性中的作用。 我们将描述BRAF诱导的BIM磷酸化的程度，确定特定的位点，并分析响应于BRAF激活的BIM磷酸化的异质性。 我们将通过正常和突变的BIM的表达以及磷酸化特异性抗血清的产生来确认这些位点。 最后，我们将使用生物化学和遗传技术来探索BRAF诱导的磷酸化抑制BIM促凋亡活性的机制。 我们相信，所提出的实验将提供一个一般的系统，用于分析BRAF在正常黑素细胞转化为转移性黑色素瘤细胞中的最早作用。 此外，这些研究将导致更好地理解BRAF和BIM在控制哺乳动物细胞凋亡中的作用。 最后，我们预计这些研究将最终导致新的治疗策略的设计和实施，以靶向转移性黑色素瘤细胞的凋亡，从而缓解这种可怕的疾病。

DESCRIPTION (provided by applicant): Mutationally activated BRAF is expressed in a wide variety of human cancers with a strikingly high prevalence in melanoma. Mutated BRAF promotes sustained activation of the RAF->MEK->ERK MAP kinase signaling pathway that in turn contributes to many of the aberrant behaviors of the melanoma cell. Using conditionally active forms of BRAF, it has been demonstrated that BRAF->MEK->ERK signaling can suppress apoptosis in a variety of mammalian cells. These data are consistent with the embryonic lethal phenotype of 5/WFnullizygous mice that display a variety of apoptotic defects. BCL-2 family proteins, comprising both pro- and anti-apoptotic members, play an essential role in the regulation of apoptosis. Hence, we find it highly provocative that we have identified a direct biochemical connection between activation of BRAF and the phosphorylation of the BH3 domain only protein BIM, a pro-apoptotic member of the BCL-2 family that plays an essential role in melanocyte apoptosis. Here we propose experiments to test the hypothesis that BRAF activation can influence apoptosis in normal melanocytes and in melanoma cells through alterations in BIM phosphorylation. To test this hypothesis we will utilize a system for the conditional activation of BRAF signaling in melanocytes. This system will be used to explore the global effects of BRAF activation in melanocytes as well as the specific effects of BRAF on apoptosis. To complement these studies, we will use genetic (RNAi) and pharmacological inhibitors of BRAF->MEK->ERK signaling or BIM expression to assess the importance of this pathway in melanocyte/melanoma cell apoptosis in response to trophic factor deprivation, matrix detachment or DNA damage. Although there is strong genetic and biochemical evidence for an important role for BRAF signaling in the initiation and progression of human melanoma, and for BIM in the regulation of melanocyte survival, we will test the hypothesis that these proteins are linked in a direct biochemical pathway. We shall elucidate the biochemical mechanism(s) by which BRAF regulates the proapoptotic effects of BIM in mammalian cells with an emphasis on the role of phosphorylation in regulating BIM expression/activity. We will characterize the extent of BRAF-induced BIM phosphorylation, identify the specific sites and analyse the heterogeneity of BIM phosphorylation in response to BRAF activation. We shall confirm these sites by expression of normal and mutated BIM and by the generation of phospho-specific antisera. Finally, we will use biochemical and genetic techniques to explore the mechanism by which BRAF-induced phosphorylation inhibits BIM's pro-apoptotic activity. We believe that the proposed experiments will provide a general system for the analysis of the earliest effects of BRAF in the conversion of normal melanocytes into metastatic melanoma cells. Moreover, these studies will lead to a better understanding of the role(s) of BRAF and BIM in controlling apoptosis in mammalian cells. Finally, we anticipate that these studies will ultimately lead to the design and implementation of new therapeutic strategies to target metastatic melanoma cells for apoptosis leading to remission of this dread disease.