Investigating the function of the non-receptor tyrosine kinase, SRMS

研究非受体酪氨酸激酶 SRMS 的功能

• 批准号: RGPIN-2017-05564
• 负责人: Lukong, Kiven
• 金额: $ 1.82万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711255
• 关键词: Investigating; function; non; receptor; tyrosine

Tyrosine kinases are enzymes that control various biological processes that underlie overall cell and organismal growth. Cell growth, division, motility, and differentiation are key examples of such processes regulated by tyrosine kinases. Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites (SRMS) is a tyrosine kinase that is conserved across many species, including humans, mice, and zebra fish. It is also the least studied and understood of the 90 known tyrosine kinases expressed in vertebrate species. SRMS is a member a non-receptor type tyrosine kinase family known as the BRK family kinases (BFKs). BRK (Breast tumor kinase, gene name PTK6) and FRK (Fyn-related kinase) are the other two proteins that belong to this family. Both PTK6 and FRK have been extensively characterized and shown to play key cellular roles in the context of cell growth and motility. The cellular and physiological roles of SRMS, on the other hand, remain largely unknown. In 2013, our group published the first biochemical study and revealed that (a) the 50 amino-acid long N-terminal region of the SRMS protein regulates its enzymatic activity and (b) Dok1 is the first bona fide cellular substrate of SRMS. Here, I propose a new program that seeks to decipher the cellular and biochemical role of SRMS by identifying the SRMS substrates/regulated proteins (phosphoproteomics) via quantitative mass spectrometry analyses. We also aim to characterize PTK6 as a SRMS. The validation and characterization of the hits is the major part of the short-term goals of the program, which will involve current and future trainees and will all be conducted in my laboratory. We will validate and characterize the SRMS targets using a myriad of molecular techniques employed in my laboratory, such as reciprocal immunoprecipitation, GST-pulldown assays, site-directed mutagenesis, in vitro kinase assays, and confocal immunofluorescence microscopy, to name a few. Therefore, here I propose to accelerate my existing research program on SRMS and thus meaningfully contribute to a career-long goal of understanding the cellular and physiological role of SRMS and how the function of SRMS differs from PTK6, FRK, and other tyrosine kinases. Our long-term goals specific to SRMS involve developing and adapting applied proteomics knowledge using high-throughput peptide-microarrays towards understanding SRMS-regulated signalosomes. We further propose to cumulatively employ these proteomics strategies towards understanding the cellular kinetics, associated signaling pathways, and mechanism of function, all in the context of investigating the cellular and physiological roles of the BFKs. Overall, these studies can be further adapted to understand how the role of SRMS impacts vertebrate biology at large.

酪氨酸激酶是控制各种生物过程的酶，这些生物过程是整个细胞和有机体生长的基础。细胞生长、分裂、运动和分化是由酪氨酸激酶调节的这些过程的关键实例。Src相关激酶缺乏C-末端调节酪氨酸和N-末端豆蔻酰化位点（SRMS）是一种酪氨酸激酶，在许多物种中是保守的，包括人类，小鼠和斑马鱼。它也是脊椎动物物种中表达的90种已知酪氨酸激酶中研究和了解最少的。SRMS是一个非受体型酪氨酸激酶家族，称为BRK家族激酶（BFK）。BRK（乳腺肿瘤激酶，基因名称PTK 6）和FRK（Fyn相关激酶）是属于该家族的另外两种蛋白质。PTK 6和FRK都已被广泛表征，并显示在细胞生长和运动的背景下发挥关键的细胞作用。另一方面，SRMS的细胞和生理作用在很大程度上仍然未知。2013年，我们的小组发表了第一项生物化学研究，并揭示了（a）SRMS蛋白的50个氨基酸长的N-末端区域调节其酶活性，（B）Dok 1是SRMS的第一个真正的细胞底物。在这里，我提出了一个新的计划，旨在破译SRMS的细胞和生化作用，通过确定SRMS底物/调节蛋白质（磷酸蛋白质组学），通过定量质谱分析。我们还旨在将PTK 6表征为SRMS。命中的验证和表征是该计划的短期目标的主要部分，这将涉及当前和未来的学员，并将在我的实验室进行。我们将验证和表征的SRMS目标使用无数的分子技术在我的实验室，如互惠免疫沉淀，GST-下拉测定，定点诱变，体外激酶测定，共聚焦免疫荧光显微镜，仅举几例。因此，在这里，我建议加速我现有的SRMS研究计划，从而有意义地有助于理解SRMS的细胞和生理作用以及SRMS的功能如何不同于PTK 6，FRK和其他酪氨酸激酶的职业生涯目标。我们的长期目标，具体到SRMS涉及开发和适应应用蛋白质组学知识，使用高通量肽微阵列对理解SRMS调节信号体。我们进一步建议累积使用这些蛋白质组学策略，以了解细胞动力学，相关的信号通路和功能机制，所有这些都是在调查BFK的细胞和生理作用的背景下。总的来说，这些研究可以进一步适应了解SRMS的作用如何影响脊椎动物生物学。