Mapping the conformation-specific interactome of signaling proteins

 描述（由申请人提供）：蛋白质是动态分子，可以采用多种构象以实现其各自的功能。虽然晶体学研究和体外生物化学测定可以对蛋白质的结构和功能提供有价值的见解，但研究活细胞中蛋白质的细胞功能仍然很困难。在加州大学弗朗西斯科分校（UCSF）的James威尔斯博士实验室担任博士后研究员期间，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，我建议使用酶标记结合定量蛋白质组学来研究动态信号蛋白BRAF激酶和KRAS GT3。KRAS-BRAF信号通路中的突变在约30%的人类癌症中发现。虽然我们了解突变激活KRAS的生化机制，但这些突变如何改变KRAS产生的蛋白质-蛋白质相互作用（其相互作用组），以及这些变化对细胞信号传导的影响仍不清楚。KRAS效应蛋白BRAF中的致癌突变通常使激酶具有组成型活性，并且不依赖于KRAS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，已经显示BRAF的小分子抑制剂可以矛盾地激活下游MAP激酶信号传导途径。这种自相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的相互作用体。该提案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肿瘤发生中KRAS和BRAF信号传导的了解，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，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。这些技能将为我提供科学基础，以实现我的长期研究目标，即了解信号蛋白的特定构象状态与其细胞功能的关系。此外，在此奖项的指导阶段，我将从我的导师威尔斯博士和我的咨询委员会的其他成员那里获得建议和培训，这将帮助我为成功的独立调查员职业生涯做好准备。在该奖项的指导阶段获得的数据将作为我作为独立调查员的第一轮赠款申请的初步数据，大大增加了我成功的可能性。完成本申请中提出的研究和培训将为我提供成功实现领导独立研究实验室的长期职业目标所需的专业知识。

 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.