Mapping the conformation-specific interactome of signaling proteins

 描述(申请人提供)：蛋白质是动态的分子，可以采用多种构象来完成各自的功能。虽然结晶学研究和体外生化分析可以为蛋白质的结构和功能提供有价值的见解，但在活细胞中研究蛋白质的细胞功能仍然是困难的。作为加州大学旧金山分校詹姆斯·威尔斯博士实验室的博士后研究员，我一直在开发酶标记技术，以研究活细胞和裂解物中的蛋白质相互作用。在这里，我建议使用酶标记和定量蛋白质组学相结合的方法来研究动态信号蛋白BRAF激酶和KRAS GTPase。在约30%的人类癌症中发现了KRAS-BRAF信号通路的突变。虽然我们了解了突变激活KRAS的生化机制，但这些突变如何改变KRAS(其互动体)的蛋白质-蛋白质相互作用，以及这些变化对细胞信号的影响仍不清楚。致癌突变的KRAS效应蛋白，BRAF，通常使该激酶结构性活跃，并独立于KRAS的调节。令人惊讶的是，催化死亡的BRAF突变体也被认为是肿瘤发生的驱动因素。此外，已有研究表明，BRAF的小分子抑制剂可以矛盾地激活下游的MAPK信号通路。这种矛盾的激活被认为是通过配体诱导的BRAF二聚体发生的，但我们不知道药物结合如何改变BRAF的相互作用组。这项提议的目标是确定突变和配体如何改变活细胞中KRAS和BRAF的相互作用组。在目标1中，我将确定用ATP竞争性抑制剂治疗后BRAF相互作用组的变化。在目标2中，我将研究临床观察到的突变对KRAS相互作用组的影响。最后，在目标3中，我将研究激酶死亡的BRAF和突变的KRAS之间的相互作用。这些目标的完成将扩大我们对KRAS和BRAF信号在肿瘤发生中的知识，并可能导致治疗KRAS和BRAF驱动的癌症的新策略。在进行拟议的研究时，我将继续利用我以前的专业知识，同时获得质谱学、蛋白质组学、系统生物学、细胞生物学和癌症生物学方面的实践培训。这些技能将为我实现我的长期研究目标提供科学基础，即了解信号蛋白的特定构象状态如何与其细胞功能相关。此外，在该奖项的指导阶段，我将从我的导师Wels博士和我的顾问委员会的其他成员那里获得的建议和培训将帮助我为作为一名独立调查人员的成功职业生涯做准备。在这个奖项的指导阶段获得的数据将作为我作为独立调查人员的第一轮拨款申请的初步数据，极大地增加了我的成功几率。完成本申请中提出的研究和培训将为我提供必要的专业知识，以成功实现我领导独立研究实验室的长期职业目标。

 DESCRIPTION (provided by applicant): Proteins are dynamic molecules that can adopt multiple conformations in order to accomplish their respective functions. While crystallographic studies and in vitro biochemical assays can give valuable insight into a protein's structure and function, studying the cellular function of a protein in living cells remains difficult. While a postdoctoral fellow in Dr. James Wells' laboratory at the University of California, San Francisco (UCSF), I have been developing enzymatic-tagging technologies to study protein interactions in living cells and lysates. Here I propose using enzymatic-tagging coupled with quantitative proteomics to study the dynamic signaling proteins BRAF kinase and KRAS GTPase. Mutations in the KRAS-BRAF signaling pathway are found in ~30% of human cancers. While we understand the biochemical mechanisms by which mutations activate KRAS, how these mutations alter the protein-protein interactions that KRAS makes (its interactome), and what effect these changes have on cellular signaling, remains unclear. Cancer-causing mutations in the KRAS effector protein, BRAF, typically render the kinase constitutively active and independent of KRAS regulation. Surprisingly, catalytically-dead BRAF mutants have also been implicated as drivers of oncogenesis. Additionally, it has been shown that small-molecule inhibitors of BRAF can paradoxically activate the downstream MAP kinase signaling pathway. This paradoxical activation is thought to occur via a ligand-induced BRAF dimer, but we do not know how drug binding alters BRAF's interactome. The goal of this proposal is to identify how mutations and ligands change the interactome of KRAS and BRAF in living cells. In Aim 1, I will identify changes to the BRAF interactome after treatment with ATP-competitive inhibitors. In Aim 2, I will study the impact of clinically observed mutations on the KRAS interactome. Finally, in Aim 3, I will study the interplay between kinase-dead BRAF and mutant KRAS. Completion of these aims will expand our knowledge of KRAS and BRAF signaling in oncogenesis and may lead to new strategies for the treatment of KRAS- and BRAF-driven cancers. While performing the proposed research I will continue to utilize my previous expertise while simultaneously gaining hands-on training in mass spectrometry, proteomics, systems biology, cell biology, and cancer biology. These skills will provide me with the scientific foundation to pursue my long-term research goal of understanding how specific conformational states of signaling proteins relate to their cellular function. In addition, the advice and training I will receive from my mentor Dr. Wels and the rest of my advisory committee during the mentored phase of this award will help me to prepare for a successful career as an independent investigator. The data acquired during the mentored phase of this award will serve as preliminary data for my initial round of grant applications as an independent investigator, greatly increasing my probability of success. Completion of the research and training proposed in this application will provide me with the expertise necessary to successfully reach my long-term career goal of leading an independent research lab.