Mechanism of Action of the TBX3 Gene in Breast Cancer

项目总结 WNT信号对胚胎发育和成人组织动态平衡至关重要，并存在异常信号 导致发育障碍和疾病，包括癌症。虽然已经知道了很多，但更深层次的 从机制上理解这一信号级联将提高我们对癌症形成的理解， 进展和转移，从而开发出更有效的治疗方法。WNT/b-连锁素 信号转导是由转录辅助激活因子b-catenin的稳定驱动的。在没有WNT配体的情况下， 胞质破坏复合体使b-连环蛋白磷酸化、泛素化并降解。在WNT在场的情况下 配体、WNT受体、FrizzledLRP6和细胞内蛋白形成一种可供选择的复合体，称为 WNT信号体。这会导致b-连环蛋白的积累和b-连环蛋白靶基因的激活。最新数据 证明在WNT配体结合时，信号体被内吞。尽管数据相互冲突 在文献中，一种共识开始出现，即依赖于笼蛋白的内吞作用 信号体导致信号体降解。这份培训方案和我的论文项目致力于 阐明正常人信号体形成、稳定和内吞作用的分子事件和动力学 在细胞和癌症中，强调对激酶的研究。在我研究生培训的前半部分，我利用了- 功能筛选确定AAK1是WNT信号的负调控因子。我证明了这一点 AAK1激活转录非依赖的负反馈环，促进LRP6内化，导致 WNT信号下调。在这些研究过程中，我们证明了AAK1促进 WNT3a后8-10小时网状蛋白接头蛋白AP2M1的磷酸化以及AAK1和AP2M1相互作用 肿瘤抑制因子WTX的作用。我的实验室之前发现WTX肿瘤抑制因子是 信号体和b-连环蛋白破坏复合体。因此，我将定义全面的WNT3a和WTX- 定量质谱法检测磷酸化蛋白质组的依赖性变化并检验WTX是否调节 通过AAK1的信号小体内吞作用。此外，已知CSNK1g调节LRP6的磷酸化，以及 信号体形成的必要步骤。CSNK1g有3种亚型，CSNK1g1/2/3，均被鉴定为未被研究的亚型 激活剂。我的初步数据显示，每种异构体在激活WNT信号和促进 LRP6内化。我剩下的研究生工作的一个主要焦点将是从功能上描述 每个CSNK1g亚型在调节WNT信号中的作用，确定全面的蛋白质-蛋白质相互作用 网络，并评估WNT驱动的磷酸蛋白质组的异构体特定变化。因为这项工作是 由于是描述性的，我预计它将在14个月内提交出版。总而言之，它的确切作用 WNT信号中的内吞作用仍不清楚，围绕其机制有许多问题(S)和 内吞作用的组成及其对信号的影响。这项工作，以及我未来的博士后工作，将提供 我在WNT信号和反馈衰减机制方面的培训和经验。

PROJECT SUMMARY WNT signaling is crucial for embryonic development and adult tissue homeostasis, with aberrant signaling resulting in developmental disorders and disease, including cancer. Although much is known, a deeper mechanistic understanding of this signaling cascade will improve our understanding of cancer formation, progression and metastasis, allowing for the development of more effective therapeutics. WNT/b-catenin signaling is driven by the stabilization of the transcriptional co-activator, b-catenin. In the absence of WNT ligand, a cytosolic destruction complex phosphorylates, ubiquitylates and degrades b-catenin. In the presence of WNT ligand, the WNT receptors, Frizzled and LRP6, and intracellular proteins form an alternative complex called the WNT signalosome. This results in b-catenin accumulation and activation of b-catenin target genes. Recent data demonstrate that upon WNT ligand engagement, the signalosome is endocytosed. Although conflicting data exist within the literature, a consensus is beginning to emerge that clathrin-dependent endocytosis of the signalosome results in signalosome degradation. This training proposal and my thesis project is devoted to elucidating the molecular events and dynamics of signalosome formation, stabilization and endocytosis in normal cells and in cancer, with an emphasis on kinases. In the first half of my graduate training, I utilized a gain-of- function screen of the kinome to identify AAK1 as a negative regulator of WNT signaling. I demonstrated that AAK1 activates a transcription independent negative feedback loop to promote LRP6 internalization, resulting in WNT signaling downregulation. In the course of these studies, we demonstrated that AAK1 promotes the phosphorylation of a clathrin adapter protein, AP2M1, 8-10 hrs post-WNT3A and that AAK1 and AP2M1 interact with the tumor suppressor, WTX. My lab previously discovered the WTX tumor suppressor as a component of the signalosome and b-catenin destruction complex. Therefore, I will define comprehensive WNT3A and WTX- dependent changes to the phosphoproteome by quantitative mass spectrometry and test whether WTX regulates signalosome endocytosis via AAK1. Additionally, CSNK1g is known to regulate phosphorylation of LRP6, an essential step for signalosome formation. CSNK1g has 3 isoforms, CSNK1g1/2/3, all identified as understudied kinases. My preliminary data suggest each isoform functions differently to activate WNT signaling and promote LRP6 internalization. A main focus for the remainder of my graduate work will be to functionally characterize the role of each CSNK1g isoform in regulating WNT signaling, define comprehensive protein-protein interaction networks and evaluate isoform specific changes to the WNT-driven phosphoproteome. Because this work is descriptive in nature, I expect it to be submitted for publication in 14 months. To summarize, the precise role of endocytosis in WNT signaling remains unclear, with numerous questions surrounding the mechanism(s) and components of endocytosis and its effects on signaling. This work, and my future postdoctoral work, will provide me training in and experience in the mechanisms of WNT signaling and feedback attenuation.