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激酶信号通路的持续激活，进而导致黑色素瘤细胞的许多异常行为。利用条件活化形式的BRAF，已经证明BRAF->MEK->ERK信号可以抑制多种哺乳动物细胞的凋亡。这些数据与显示各种凋亡缺陷的5/ wfnullizyous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，在细胞凋亡的调控中发挥重要作用。因此，我们发现BRAF的激活与BH3结构域蛋白BIM的磷酸化之间存在直接的生化联系，这是非常具有挑战性的，BH3结构域蛋白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，我们提出实验来验证BRAF激活可以通过改变BIM磷酸化影响正常黑色素细胞和黑色素瘤细胞的凋亡的假设。为了验证这一假设，我们将利用一个系统来条件激活黑素细胞中的BRAF信号。该系统将用于探索BRAF在黑素细胞中激活的全局效应以及BRAF对细胞凋亡的特异性作用。为了补充这些研究，我们将使用BRAF->MEK->ERK信号或BIM表达的遗传（RNAi）和药理抑制剂来评估该途径在营养因子剥夺、基质脱离或DNA损伤的黑素细胞/黑色素瘤细胞凋亡中的重要性。尽管有强有力的遗传和生化证据表明BRAF信号在人类黑色素瘤的发生和发展中发挥重要作用，BIM在黑素细胞存活的调节中发挥重要作用，但我们将测试这些蛋白质在直接生化途径中联系的假设。我们将阐明BRAF调节哺乳动物细胞中BIM的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磷酸化程度，确定具体位点，并分析响应BRAF激活的BIM磷酸化的异质性。我们将通过正常和突变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，我们将使用生化和遗传技术来探索braf诱导的磷酸化抑制BIM促凋亡活性的机制。我们相信，所提出的实验将为分析BRAF在正常黑色素细胞转化为转移性黑色素瘤细胞中的早期作用提供一个通用系统。此外，这些研究将有助于更好地理解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设计和实施针对转移性黑色素瘤细胞凋亡的新治疗策略，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.