CYTOSKELETAL REGULATION BY SRC64/TEC29 KINASES

SRC64/TEC29 激酶对细胞骨架的调节

• 批准号: 6351345
• 负责人: MICHAEL A SIMON
• 金额: $ 23.75万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351345
• 关键词: Drosophilidae; actins; cell cell interaction; cytoskeleton; enzyme activity; enzyme induction /repression; enzyme substrate; histogenesis; intermolecular interaction; laboratory rabbit; molecular cloning; oogenesis; ovary; phosphoproteins; protein tyrosine kinase; site directed mutagenesis

Members of the SRC family of non-receptor protein tyrosine kinases (SFKs) play crucial roles in the regulation of cellular growth, differentiation, and morphology. Studies in vertebrates have shown that inappropriate activation of SFKs can lead to oncogenic transformation of cells, while the absence of SFKs has been associated with defects in signaling by the B and T cell antigen receptors and with defects in bone resorption (osteopetrosis). Among the important cellular consequences of altered SFK function are defects in the cellular actin cytoskeleton. The aim of the proposed research is to investigate how SFKs participate in cytoskeletal regulation. We are addressing this issue by studying the action of a particular SFK, the product of the Drosophila Src64 gene. We have chosen to study Src64 because of the ability to perform genetic studies that would be either difficult or impossible to conduct in vertebrate organisms. Our goal is to combine these genetic studies with extensive biochemical analysis in order to understand Src64 function. In our previous work, we identified mutations that inactivated the Src64 gene. We showed that the lack of Src64 function has a surprisingly specific phenotype: Src64 mutant animals are fully viable, but the females are partially sterile. Our analysis of this phenotype revealed that it is associated with defects in the morphogenesis of specialized actin cytoskeletal structures, called ring canals, that form at the cytoplasmic bridges connecting the developing oocyte and its adjacent cluster of nurse cells. We then genetically identified the Tec29 protein as a major target of SRC64 action. ThC29 is a member of a family of tyrosine kinases that includes vertebrate proteins such as Bruton's tyrosine kinase, whose absence in humans leads to X-linked agammaglobulinemia. We showed that TEC29 is essential for normal ring canal morphogenesis and is recruited to the growing ring canals in response to SRC64 activity. The goals of the proposed research are to further characterize the role of SRC64 and TBC29 in actin cytoskeletal regulation by: 1) investigating the role of SRC64 and ThC29 using domain specific mutagenesis, 2) investigating the biochemical basis for SRC64 regulation of TEC29, 3) identifying and characterizing substrates of TEC29, 4) cloning and analyzing E(Src64)2B, another potential target of SRC64, and 5) conducting further genetic screens to identify additional components of the SRC64/TEC29 pathway.

SRC家族的非受体蛋白酪氨酸激酶（SFKs）在细胞生长、分化和形态的调控中起着至关重要的作用。在脊椎动物中的研究表明，SFKs的不适当激活可导致细胞的癌性转化，而SFKs的缺失与B细胞和T细胞抗原受体的信号传导缺陷以及骨吸收缺陷（骨质疏松症）有关。SFK功能改变的重要细胞后果之一是细胞肌动蛋白细胞骨架的缺陷。本研究的目的是研究sfk如何参与细胞骨架调节。我们正在通过研究果蝇Src64基因的产物SFK的作用来解决这个问题。我们之所以选择研究Src64，是因为它有能力进行在脊椎动物生物中很难或不可能进行的遗传研究。我们的目标是将这些遗传研究与广泛的生化分析相结合，以了解Src64的功能。在我们之前的工作中，我们发现了使Src64基因失活的突变。我们发现缺乏Src64功能具有令人惊讶的特异性表型：Src64突变动物完全可存活，但雌性部分不育。我们对这种表型的分析表明，它与称为环管的特殊肌动蛋白细胞骨架结构的形态发生缺陷有关，环管形成于连接发育中的卵母细胞及其邻近的护理细胞簇的细胞质桥。然后，我们从基因上鉴定出Tec29蛋白是SRC64作用的主要靶点。ThC29是酪氨酸激酶家族的一员，该家族包括脊椎动物蛋白，如布鲁顿酪氨酸激酶（Bruton’s tyrosine kinase），人类缺乏这种蛋白会导致x连锁无球蛋白血症。我们发现TEC29对于正常的环管形态形成至关重要，并且在SRC64活性的作用下被招募到生长的环管中。本研究的目标是通过以下方式进一步表征SRC64和TBC29在肌动蛋白细胞骨架调节中的作用：1)利用结构域特异性诱变研究SRC64和ThC29的作用，2)研究SRC64调控TEC29的生化基础，3)鉴定和表征TEC29的底物，4)克隆和分析SRC64的另一个潜在靶点E(SRC64)2B, 5)进行进一步的遗传筛选以鉴定SRC64/TEC29通路的其他组分。