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

酪氨酸激酶是一种控制各种生物过程的酶，这些过程构成了整个细胞和生物生长的基础。细胞的生长、分裂、运动和分化是由酪氨酸激酶调节的这些过程的关键例子。SRC相关蛋白是一种缺乏C-末端调节性酪氨酸和N-末端肉豆蔻酸化位点(SRMS)的酪氨酸激酶，在包括人类、小鼠和斑马鱼在内的许多物种中都是保守的。在已知的90种在脊椎动物中表达的酪氨酸激酶中，它也是研究和了解最少的。SRMS是一种非受体类型的酪氨酸激酶家族成员，被称为BRK家族激酶(BFK)。BRK(乳腺肿瘤激酶，基因名为PTK6)和Frk(Fyn-Related Kinase)是属于该家族的另外两种蛋白质。PTK6和Frk都已被广泛研究，并被证明在细胞生长和运动环境中扮演关键的细胞角色。另一方面，SRMS的细胞和生理作用在很大程度上仍不清楚。2013年，我们的研究小组发表了第一项生化研究，发现(A)SRMS蛋白的50个氨基酸的N末端区域调节其酶活性，(B)DOK1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，试图通过定量质谱分析识别SRMS底物/受调控的蛋白质(磷蛋白质组学)来破译SRMS的细胞和生化作用。我们还打算将PTK6描述为一种SRMS。HITS的验证和表征是该项目短期目标的主要部分，该项目将涉及当前和未来的学员，所有工作都将在我的实验室进行。我们将使用我的实验室中使用的大量分子技术来验证和表征SRMS靶标，例如相互免疫沉淀、GST下拉试验、定点突变、体外激酶分析和共聚焦免疫荧光显微镜，仅举几例。因此，在这里，我建议加快我现有的SRMS研究计划，从而有意义地为了解SRMS的细胞和生理作用以及SRMS的功能与PTK6、Frk和其他酪氨酸激酶的不同而做出有意义的贡献。我们特定于SRMS的长期目标包括使用高通量多肽微阵列开发和调整应用蛋白质组学知识，以了解SRMS调节的信号小体。我们进一步建议在研究BFK的细胞和生理作用的背景下，累积使用这些蛋白质组学策略来理解细胞动力学、相关的信号通路和功能机制。总体而言，这些研究可以进一步调整，以了解SRMS的作用如何影响整个脊椎动物生物学。

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.