臺灣博碩士論文加值系統

蛋白質酪胺酸激酶包括受體型蛋白質酪胺酸激酶與非受體型蛋白質酪胺酸激酶是一群重要的訊息傳遞調控者，他們調控細胞生長，分化與發育。Tec非受體型蛋白質酪胺酸激酶與細胞激素受體，淋巴球表面抗原，G蛋白結合受體及integrin 的細胞傳遞有關。大多數的Tec非受體型蛋白質酪胺酸激酶表現在造血細胞，然而其中一員Etk/Bmx ，主要表現在上皮細胞與內皮細胞且其在這些細胞中的訊息傳遞及生物功能的研究有限。在我們探討Etk的活化因子時，發現EGF和VEGF皆能活化Etk。在HeLa及MDA-MB-468細胞株中，Etk可以在EGF的刺激下被磷酸化。並且在MDA-MB-468細胞株中，大量表達此蛋白會透過活化Stat1而增加EGF誘導的細胞凋亡。相反的，dominant-negative Etk (Ek)能抑制Stat1活性進而降低EGF誘導的細胞凋亡。再者，沒有EGF處理下，持續活化Etk能促使MDA-MB-468細胞凋亡。這些結果證實Etk是EGFR下游的一訊息傳遞分子並在EGF誘導的細胞凋亡中扮演重要角色。在VEGF的研究中，我們第一個發現在人類臍靜脈內皮細胞，突變型Etk可抑制VEGF誘導的血管生成作用。免疫螢光染色實驗也證實Ek降低VEGF誘導細胞與細胞間的分離，顯示Etk可調節內皮細胞間的連結。體外與體內蛋白結合實驗皆證實Etk直接與-catenin結合並能磷酸化-catenin。 此外，Ek降低-catenin磷酸化並防止-catenin離開細胞間的連結處。Etk抑制劑LFM-A13，也能抑制-catenin磷酸化及VEGF的在內皮細胞的作用。這些發現顯示Etk藉由與-catenin結合並磷酸化-catenin來調控VEGF所誘導的生物現象。綜合上述實驗結果，我們證實Etk的確在EGF誘導的細胞凋亡與VEGF誘導的細胞移行的訊息傳遞中扮演非常重要的角色。

Protein tyrosine kinases, including receptor protein-tyrosine kinases and non-receptor protein tyrosine kinases, are important regulators of signal transduction for activation of cell growth, differentiation and development. Tec family of non-receptor protein kinases are involved in the intracellular signaling mechanisms of cytokine receptors, lymphocyte surface antigens, heterotrimeric G-protein-coupled receptors and integrin molecules in various cell types. While most of Tec family kinase proteins are expressed in hematopoietic cells, Etk/Bmx, a member of the Tec family protein kinases, is mainly expressed in epithelial and endothelial cells. Very little is known about Etk’s signaling networks and biological functions in these cells. We have explored different stimuli in activating Etk activity in epithelial and endothelial cells. Among them, EGF and VEGF were found to activate Etk. More specifically, Etk could be phosphorylated and activated by EGF in HeLa and MDA-MB-468 cells, and overexpression of Etk enhanced EGF-induced cell apoptosis of MDA-MB-468 cells. In addition, the Etk-enhanced cell apoptosis in MDA-MB-468 cells is associated with the Stat1 activation. In contrast, a dominant-negative Etk could abrogate Stat1 activation and EGF-induced apoptosis in MDA-MB-468 cells. Furthermore, a conditionally active Etk could induce Stat1 activity and confer cell apoptosis in MDA-MB-468 cells in the absence of EGF treatment. These findings suggest that Etk functions as a downstream signal molecule of EGF receptor and plays an important role in EGF-induced apoptotic signaling. In prior VEGF studies, we first demonstrated that a dominant-negative Etk inhibited the capillary-like tube formation of human umbilical vein endothelial cells on the Matrigel and reduced endothelial cell migration induced by VEGF. Immunofluorescence studies revealed that the dominant-negative mutant of Etk abolished VEGF-induced cell-cell dissociation, suggesting that Etk is involved in regulating adherens junction of endothelial cells. In vitro and in vivo binding studies showed that Etk directly interacts and phosphorylates -catenin. Moreover, a kinase deletion mutant of Etk, blocks the phosphorylation of -catenin and prevents the dissociation of -catenin from the adherens junction in response to VEGF. Finally, LFM-A13, a compound inhibiting Etk activity, diminishes the tyrosine phosphorylation of -catenin and cell mobility induced by VEGF. These findings implicate a crucial role of Etk in VEGF-induced cellular events via the interaction and phosphorylation of -catenin. Together, Etk plays a pivotal role in mediating the intracellular signaling pathways and functions of EGF-induced apoptosis and VEGF-induced cell migration.

Table of Content ……………………………………………………………………………….1
中文摘要……………………………………………………………………………………….6
Abstract…………………………………………………………………………………………7
Chapter I General introduction………………………………………………………………..9
PH domain
TH (BH and PR) domain
SH3 domain
SH2 domain
Kinase domain
Activation of Tec kinases
EGF signaling and VEGF signaling
Cadherins junctions
Chapter II Role of tyrosine kinase Etk/Bmx in EGF-induced apoptosis of MDA-MB-468 breast cancer cells……………………………………………………………………………...19
Abstract……………………………………………………………………………………….20
Introduction…………………………………………………………………………………...21
Results…………………………………………………………………………………………23
EGF stimulates tyrosine phosphorylation of Etk
Etk potentiates EGF-induced apoptosis of MDA-MB-468 cells
Etk potentiates EGF-induced Stat1 activation
A dominant negative mutant of Etk inhibits
Conditionally activated Etk induces both Stat1 activation and cell apoptosis
Discussion…………………………………………………………………………………….28
Materials and methods………………………………………………………………………..32
Plasmid construction
Cell culture, Western blot analysis, transient transfection and luciferase assays
Flow cytometric analysis of apoptosis
Electrophoretic mobility shift assay (EMSA)
Chapter III The role of Etk/Bmx in vascular endothelial growth factor-induced endothelial cell-cell dissociation and migration……………………………………………………………34
Abstract......................................................................................................................................35
Introduction……………………………………………………………………………………36
Results........................................................................................................................................39
Dominant-negative Etk abrogates VEGF-induced capillary-like formation and cell migration
Etk associates with -catenin in HUVECs
The SH3 and SH2 domains of Etk mediates the association with -catenin
Etk stimulates tyrosine phosphorylation of -catenin
Dominant-negative Etk blocks VEGF-induced tyrosine phosphorylation of -catenin
Dominant-negative Etk blocks VEGF-induced dissociation of -catenin from adherens junctions
Etk kinase activity is crucial for the tyrosine phosphorylation of -catenin and cell mobility induced by VEGF treatment
Discussion..................................................................................................................................44
Materials and methods………………………………………………………………………...47
Plasmid and recombinant adenovirus construction
Cell culture
Cell migration assay and capillary-like tube formation
In vitro GST pull down assay
Immunoprecipitation and immunoblotting analyses
Immunofluorescence analysis
Chapter IV General discussion…………………………………………………………………50
Figures..........................................................................................................................................57
Chapter I General introduction.............................................................................................57
Fig. 1 Tec kinase activation
Fig. 2 Cadherens junctions
Chapter II Role of tyrosine kinase Etk/Bmx in EGF-induced apoptosis of MDA-MB-468 breast cancer cells................................................................................................................59
Fig. 1A+1B EGF stimulates tyrosine phosphorylation of Etk
A. HeLa cells
B. MDA-MB-468 cells.
Fig. 1C EGF induces the phosphorylation of ectopically expressed Etk
Fig. 2 Etk potentiates EGF-induced apoptosis
Fig. 3A Etk potentiates EGF-induced Stat1 activation
Fig. 3B Etk potentiates the effect of EGF on the Stat1-mediated promoter and p21 promoter
Fig. 4A + 4B
A. Dominant negative Etk inhibits EGF-induced Stat1 activation and apoptosis.
B. EtkK blocked EGF-induced Stat1 activation
Fig. 4C EtkK abrogated EGF-induced apoptosis.
Fig. 5 Activation of Etk kinase activity is sufficien t to induce the Stat1 activity and cell apoptosis
Fig. 5B Conditionally activated Etk induces Stat1 activity
Fig. 5C Conditionally activated Etk stimulates Stat1-mediated promoter and p21 promoter
Fig. 5D Conditionally activated Etk induces cell apoptosis
Chapter III The role of Etk/Bmx in vascular endothelial growth factor-induced endothelial cell-cell dissociation and migration…………………………………………………………71
Fig. 1A Role of Etk in VEGF-induced capillary-like tubule formation and cell migration of HUVECs
Fig. 1B Wild-type Etk promotes, while a dominant-negative EtkK inhibits, VEGF-induced cell migration of HUVECs.
Fig. 2A Etk associates with b—catenin in HUVECs.
Fig. 2B Etk and b-catenin associate together in HUVECs
Fig. 3 Multiple domains of Etk are involved in the interaction with-catenin
Fig. 3C Interaction of -catenin with SH3 and SH2 domains of Etk in vitro
Fig. 4A Etk is an upstream kinase of-catenin in VEGF signaling pathway
Fig. 4B Etk stimulates tyrosine phosphorylation of -catenin in Cos-1 cells
Fig. 4C EK downregulates VEGF-induced tyrosine phosphorylation of -catenin in HUVECs
Fig. 5 Effect of Etk on the distribution of cadherin-associated protein complexes in HUVECs in response to VEGF
Fig 6A Etk kinase activity is crucial for VEGF-induced tyrosine phosphorylation of -catenin and cell migration of HUVECs
Fig 6B Wound healing was observed after 16 hours of treatment with 20 ng/ml VEGF and 25 mM LFM-A13
Fig. 7 Schematic illustration of the role of Etk in VEGF-induced angiogenic pathway in endothelial cells
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