CYTOSKELETAL REGULATION BY SRC64/TEC29 KINASES

SRC家族中的非受体蛋白酪氨酸激酶(SFK)在调节细胞生长、分化和形态方面起着至关重要的作用。脊椎动物的研究表明，SFK的不适当激活可以导致细胞的致癌转化，而SFK的缺失与B和T细胞抗原受体的信号传递缺陷以及骨吸收缺陷(骨化症)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。这项拟议研究的目的是调查SFK如何参与细胞骨架的调节。我们正在通过研究一种特殊的SFK的作用来解决这个问题，SFK是果蝇Src64基因的产物。我们之所以选择研究src64，是因为我们有能力进行在脊椎动物中难以或不可能进行的遗传学研究。我们的目标是将这些遗传学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定表型：src64突变动物完全存活，但雌性动物部分不育。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的形态发生缺陷有关，这种结构被称为环管，形成于连接发育中的卵母细胞及其邻近的哺育细胞簇的细胞质桥梁。然后，我们从基因上确定Tec29蛋白是SRC64作用的主要靶点。ThC29是酪氨酸激酶家族的成员之一，该家族包括脊椎动物蛋白，如Bruton‘s酪氨酸激酶，该蛋白在人类中的缺失会导致X连锁无丙种球蛋白血症。我们发现TEC29对正常的环管形态发生是必不可少的，并且在SRC64活性的响应下被募集到生长的环管中。本研究的目标是通过以下方式进一步研究SRC64和TBC29在肌动蛋白细胞骨架调控中的作用：1)通过区域特异性突变研究SRC64和ThC29的作用；2)研究SRC64调控TEC29的生化基础；3)鉴定和鉴定TEC29的底物；4)克隆和分析SRC64的另一个潜在靶标E(Src64)2B；以及5)进行进一步的遗传筛选，以确定SRC64/TEC29途径的其他成分。

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.