Live Cell Isoform-specific Akt Analyses

该项目正在开发能够区分接近的异构体的活细胞激酶分析方法。AKT1和 Akt2是我们最初的激酶模型。这两个蛋白有很高的序列同源性，但却神秘地显示出来 发育、糖尿病等方面的功能差异。我们的目标是开发荧光活细胞亚型- 特异性Akt活性的量化，并确定Akt1和Akt2底物是否具有不同的特异性 和/或偏好是一种潜在机制。这些方法具有广泛的适用性。该项目将培育 许多本科生研究人员的成长到他们生物医学职业生涯的下一个阶段。 通过将凯文·肖卡特博士开发的成熟的化学遗传学方法与 细胞不透膜的块状类似物N6-苄基-三磷酸腺苷-g-S(A*TP-g-S)的纳米递送 活细胞中的特定底物标记，即区分感兴趣的激酶反应与 人类基因组中编码的那些&gt；500激酶--这是活细胞“激酶分析”的先决条件。简单地说， 根据Shokat方法，Akt1/2网守Met(M)突变为Gly(G)以扩大ATP 结合口袋以适应A*TP；即创建Akt1M-G和Akt2M-G突变体。我们向大家展示了 只有Akt1/2M-G能有效利用传递的A*TP-g-S，这是因为腺嘌呤部分较大，而硫代磷酸-g-G- 标记它们的底物。目前，A*TP正在用MANT或TNP荧光染料标记。排他 AKT1/2M-G与纳米粒递送的MANT-/TNP-A*TP结合将激活其荧光，进而 量化Akt1/2ATP结合，从而在完整的活细胞中的酶活性；即，活细胞量化 AKT1/2激酶活性。我们还通过基因组编辑产生突变，因此Akt1/2M-G基因 停留在本地基因组环境中。我们的目标是：目标1：荧光活细胞 通过表达载体定量Akt-ATP结合活性；目标2：使该方法适用于初级编辑 带有Akt1或Akt2ATP结合口袋的人细胞，即Akt1M→G细胞和Akt2M→ 目标3：鉴定和区分底物的Akt1和Akt2谱。 意义：我们正在开发一种广泛适用于活细胞激酶研究的系统。激活酶-底物 通过活细胞中底物标记的关系分析将被建立。我们的荧光活细胞激活酶 化验将绕过人工底物的使用，从而能够区分同源激酶等 Akt1和Akt2等亚型。这一结果将为研究蛋白激酶建立一个新的、通用的范式。 AKT1和Akt2长期以来一直是药物和治疗开发的目标。识别他们各自的身份 底物光谱将有助于指导相关人类的进一步开发和改进工作 疾病。此外，该项目为学生提供了一个很好的跨学科平台来整合 课堂教育和研究活动。

This project is developing live-cell kinase analysis methods that can distinguish close isoforms. Akt1 and Akt2 are our initial kinase model. The two kinases share high sequence homology, but mysteriously display functional difference in development, diabetics and et al. We aim to develop fluorescent live-cell isoform- specific Akt activity quantification, and to determine whether different Akt1 and Akt2 substrate specificity and/or preference are an underlying mechanism. The methods are widely applicable. The project will nurture the growth of many undergraduate researchers to their next stage of their biomedical career. By combining the well-established chemical-genetic method developed by Dr. Kevan Shokat and nanoparticle-delivery of the cell-impermeable bulky analog N6-benzyl-ATP-g-S (A*TP-g-S), we achieved specific substrate tagging in live cells, that is, the ability to distinguish the kinase reaction of interest from those of the > 500 kinases encoded in the human genome – a prerequisite for live-cell “kinase assay”. Briefly, according to the Shokat method, Akt1/2 gatekeeper Met (M) was mutated to Gly (G) to enlarge the ATP binding pockets to accommodate A*TP; i.e., creation of the Akt1M-G and Akt2M-G mutants. We showed that only Akt1/2M-G could efficiently use delivered A*TP-g-S due to the bulkier adenine moiety, and thiophosphate- tag their substrates. Currently, A*TP is being tagged with the MANT or TNP fluorogenic dyes. Exclusive Akt1/2M-G binding to nanoparticle-delivered MANT-/TNP-A*TP will activate their fluorescence, which in turn quantify Akt1/2 ATP-binding, and thus enzymatic, activities in intact live cells; that is, live-cell quantification of Akt1/2 kinase activities. We are also generating the mutations via genome editing, so that Akt1/2M-G genes stay in native genomic context. Our aims to accomplish the goals are: Aim 1: Fluorogenic live-cell quantification of Akt ATP binding activities via expression vectors; Aim 2: Adapting the method to prime-edited HCT116 human cells with enlarged Akt1 or Akt2 ATP binding pockets, i.e., the Akt1M→G cells and the Akt2M→ G cells; and Aim 3: To identify and differentiate the Akt1 and Akt2 spectrums of substrates. Significance: We are developing a widely applicable system for live-cell kinase studies. Kinase-substrate relationship analysis via substrate tagging in live cells will be established. Our fluorogenic live-cell kinase assay will bypass usage of artificial substrates to enable, among others, distinguishing homologous kinase isoforms such as Akt1 and Akt2. The results will set a new and general paradigm for studying protein kinases. Akt1 and Akt2 have long been targeted for drug and therapeutic development. Identification of their respective spectrum of substrates will help guide further development and improvement efforts for relevant human diseases. Additionally, the project provides an excellent interdisciplinary platform for students to integrate classroom education and research activities.