CYTOSKELETAL REGULATION BY SRC64/TEC29 KINASES

SRC64/TEC29 激酶对细胞骨架的调节

• 批准号: 6628904
• 负责人: MICHAEL A SIMON
• 金额: $ 25.19万
• 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/6628904
• 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（SFK）的成员在细胞生长、分化和形态的调节中起着至关重要的作用。对脊椎动物的研究表明，SFKs的不适当活化可导致细胞的致癌转化，而SFKs的缺乏与B和T细胞抗原受体的信号传导缺陷以及骨吸收缺陷（骨硬化症）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。本研究的目的是探讨SFKs如何参与细胞骨架的调控。我们正在解决这个问题，通过研究一个特定的SFK，果蝇Src 64基因的产物的行动。我们之所以选择研究Src 64，是因为它能够进行在脊椎动物中难以或不可能进行的遗传研究。我们的目标是联合收割机这些遗传研究与广泛的生化分析，以了解Src 64功能。在我们之前的工作中，我们鉴定了使Src 64基因失活的突变。我们发现，Src 64功能的缺乏具有令人惊讶的特定表型：Src 64突变动物是完全可行的，但女性部分不育。我们对这种表型的分析表明，它与特殊的肌动蛋白细胞骨架结构（称为环形通道）的形态发生缺陷有关，环形通道形成于连接发育中的卵母细胞及其相邻的护理细胞簇的细胞质桥处。然后，我们在遗传学上鉴定了Tec 29蛋白作为SRC 64作用的主要靶标。ThC 29是酪氨酸激酶家族的成员，该家族包括脊椎动物蛋白，例如布鲁顿酪氨酸激酶，其在人类中的缺失导致X连锁无丙种球蛋白血症。我们发现，TEC 29是必不可少的正常环管形态发生，并招募到不断增长的环管响应SRC 64的活动。拟议研究的目标是通过以下方式进一步表征SRC 64和TBC 29在肌动蛋白细胞骨架调节中的作用：1）使用结构域特异性诱变研究SRC 64和ThC 29的作用，2）研究SRC 64调节TEC 29的生化基础，3）鉴定和表征TEC 29的底物，4）克隆和分析E（Src 64）2B，SRC 64的另一个潜在靶标，和5）进行进一步的遗传筛选以鉴定SRC 64/TEC 29途径的其他组分。