A PTPome-wide Analysis of Drosophila Non-transmembrane Protein Tyrosine Phosphatases Unveils the Requirement of dHD-PTP in Development

蛋白質酪氨酸去磷酸酶(PTP)是一群受到嚴密調控的酵素， 並且在各種不同的過程中與蛋白質酪氨酸磷酸酶協同控制蛋白質的磷酸化。目前許多的證據指出，在發育過程當中，蛋白質磷酸化的調控扮演重要的角色。為了了解PTP在發育過程中的功能，我們使用果蠅的PTPome 作為研究PTP發育功能的一個模式。之前的研究根據蛋白質序列相似度分析發現果蠅基因體中存在有十五個可能的PTP。其中，七個是類似受體型的PTP，而八個則是非受體型的PTP。近年的研究都針對類似受體型PTP在神經發育過程中所扮演的角色來進行分析，但是非受體型PTP的功能仍然有待理解。了要了解發育過程中這些非受體型PTP的生物功能，我有系統的分析了這些PTP 基因的表現式樣以及他們的去磷酸生化活性。我也同時建立了可以表現這些PTP全長cDNA以及雙股RNA以利用RNAi的方式降解內生性PTP的基因轉植果蠅。用原位雜交的方式，我發現許多的非受體型PTP都高度的表現在胚胎時期的中樞神經系統，而相較之下在三齡幼蟲時期的imaginal disc中只有低度且廣泛的表現。另外，利用gain-of-function 和 loss-of-function 的分析，我發現其中一個含有 BRO1 domain 的PTP，dHD-PTP，在翅膀表皮細胞形態發育中是一個不可或缺的蛋白質。了進一步研究dHD-PTP的功能，利用免疫螢光染色，我發現dHD-PTP蛋白質位於早期和晚期的endosomes。 令人驚訝的，不論是過度表現或是降解dHD-PTP的表現量都會影響到endosomes的外觀形態，這個結果意味dHD-PTP可能在endoomes 結構的生成過程中扮演調控的角色。外，過度表現 cyclineE，一個調控細胞週期進行的基因，可以部份的拯救因為dHD-PTP loss-of-function所產生的翅膀缺陷。而在抑制dHD-PTP表現量的mosaic clones中會有過度不正常的細胞死亡現象，表示dHD-PTP有可能參與在細胞生長以及細胞生存的的調節之中。由遺傳分析，我發現dPax， 一個focal adhesion adaptor 蛋白質，在翅膀表皮細胞的發育過程當中與dHD-PTP有遺傳上的交互作用 。而言之，我證明dHD-PTP在發育過程中對於胞吞作用以及細胞黏著的調控是一個重要的蛋白質。dHD-PTP可能是藉由其在胞吞作用所扮演的功能來調節與細胞黏著相關的蛋白質的交通。結合一開始針對果蠅非受體型PTP基本的分析，我的研究應該對於這些PTP在發育過程中的功能有其啟發性的貢獻。Protein tyrosine phosphatases (PTPs) are a group of tightly regulated enzymes in coordination with protein tyrosine kinases to control protein phosphorylation during various cellular processes. Accumulating evidence has indicated that regulation of tyrosine phosphorylation plays a crucial role during development. Drosophila PTPome is chosen as a model to define the developmental function of PTPs. Based on sequence similarity, fifteen putative PTPs in Drosophila genome are identified. Among them, seven of them are receptor like PTPs, and eight are predicted to be non-transmembrane PTPs. While much research has been devoted to the role of receptor-like tyrosine phosphatases in neural development, the function of non-transmembrane PTPs remains to be elucidated. o investigate the biological function of these non-transmembrane PTPs during development, I have systematically analyzed the gene expression pattern and phosphatase activity of these PTPs. Moreover, transgenic flies carrying wild type cDNA or double-stranded RNA of each non-transmembrane PTP were generated. in situ hybridization revealed that many of these PTPs are highly expressed in embryonic CNS and exhibited relatively low and ubiquitous expression in third instar larval imaginal discs. In addition, loss-of-function and gain-of function analysis revealed that dHD-PTP, a BRO1 domain containing protein tyrosine phosphatase, is required for wing morphogenesis. Immunofluorescence showed that dHD-PTP is localized at both early and late endosomes. Strikingly, both downregulation and overexpression of dHD-PTP affected the size of endosomes, suggesting a regulatory role of dHD-PTP in the biogenesis of endosomal structures. n addition, I found that overexpression of cyclinE, a gene known for it role in cell cycle progression, could partially rescue dHD-PTP loss-of-function defects in developing wing. This, along with the observation of ectopic cell death in dHD-PTP loss-of-function mosaic clones suggests possible involvements of dHD-PTP in cell proliferation and cell survival. Genetic analysis also found that dPax, a focal adhesion adaptor protein, genetically interacts with dHD-PTP during wing epithelia development.n summary, I demonstrate that dHD-PTP could function as an essential protein in regulation of endocytosis and cell adhesion during development. dHD-PTP might be involved in the control of cell adhesion by its ability to regulate the trafficking of adhesion-related molecules. Combined with characterization of other non-transmembrane PTPs, my studies should shed lights on the role of these non-transmembrane PTPs in development.Table of Contents index of Figures and Tables ivbstract in Chinese vibstract viihapter One: Introduction 1. Protein tyrosine phosphatases 1I. Regulation of protein tyrosine phosphatases 4II. Biological functions of protein tyrosine phosphatases 5V. Strategies in studying the biological functions of protein tyrosine phosphatases 8. Protein tyrosine phosphatases in Drosophila Genome 9I. Functions of receptor-like protein tyrosine phosphatases in Drosophila Development 10II. Functions of non-transmembrane protein tyrosine phosphatases during Drosophila Development 13III. Genomic approach in studying biological function of non-transmembrane PTPs in Drosophila Genome 15hapter Two: Materials and Methods 17. Bioinformatics 17I. Fly Strains and Genetics 17II. DNA manipulation and Molecular cloning 17V. Generation of Transgenic Fly 18. dsRNA Generation 18I. Cell culture, Transfection and RNAi 19II. Preparation of Cultured Cell Lysates and Fly Lysates 20III. Estimation of Protein Concentration 20X. Immunohistochemistry and Immunofluorescence of Cultured Cells 20. Western Blotting 21I. Immunoprecipitation 22II. Whole mount in situ Hybridization 22III. In Gel Phosphatase Activity Assay 24IV. pNPP Phosphatase Assay 25V. BrdU Incorporation Assay 26VI. Endocytic Uptake Assay 26VII. Quantification of Dissociation and Migration Defects of Border Cells 27VIII. Antibody Generation 27hapter Three: Results 28. Characterization of protein tyrosine phosphatases in Drosophila genome 28I. Phosphatase catalytic inferences from multiple sequence alignment of Drosophila PTP genes 28II. Expression profiles of catalytic active tyrosine phosphatases revealed by in gel phosphatase assay 29V. in vitro Phosphatase assays showed possible catalytically inactive PTPs 29. Expression patterns of Drosophila PTP genes during embryogenesis and in third instar larval imaginal discs 30I. Functional inference from transgenic flies of non-transmembrane PTPs 32II. dHD-PTP is required for wing morphogenesis during development 33III. Characterization of conserved domain structure of dHD-PTP 34X. Generation of dHD-PTP antibody and validation of dHD-PTP transgenic lines 36. dHD-PTP is an essential protein required for normal development 36I. dHD-PTP expression profile during development 37II. Endogenous dHD-PTP is localized in endosomal vesicles 38III. dHD-PTP overexpression causes enlarged endosomes 38IV. Central Proline Rich Region (PRR) of dHD-PTP is required and sufficient for vesicle targeting 39V. Down regulation of dHD-PTP causes multiple enlarged endosomes 39VI. dHD-PTP loss-of -function defects may be partly caused by defective cell proliferation 40VII. Integrin loss-of-function related phenotype in ectopic expression of dHD-PTP in posterior wing 41VIII. Genetic interaction of dHD-PTP with dPax 42IX. Collective cell migration defects in dHD-PTP loss-of-function and gain-of-function lines 43hapter Four: Discussion 47. Function of catalytic inactive non-transmembrane PTPs 47I. Functional studies of HD-PTP 47II. Endocytic function of dHD-PTP 49V. Cell proliferation function of dHD-PTP has not been clearly understood 50. dHD-PTP in regulation of cell adhesion 51I. A role for dHD-PTP in collective cell migration 52hapter Five: References 10