乳癌激酶 (Breast tumor kinase, Brk) 是屬於Src-like酪氨酸激酶家族中的一員。其具有SH3，SH2及酪氨酸激酶催化區塊。一開始是在人類具有高度轉移性乳癌細胞中被發現，且其高度表現在乳癌以及許多其他的癌症細胞中。然而乳癌激酶參與在癌症形成的分子機制仍需要釐清。至今，乳癌激酶過量表現被發現會造成乳腺上皮細胞對表皮生長激素 (EGF) 敏感度上升。在本篇論文中，我們產製了同時可以辨識外生及內生乳癌激酶的抗體。而這支抗體也可以免疫沉澱下外生或內生的乳癌激酶。此外，在本篇論文中，我們發現了p190RhoGAP為乳癌激酶的新受質並更進一步確認其主要受到磷酸化作用的位置為酪氨酸1105。此外，我們也發現了乳癌激酶參與在由表皮生長激素引起的p190RhoGAP的酪氨酸1105磷酸化中。這樣的磷酸化會促進p190RhoGAP在表皮生長激素刺激之下被帶到細胞膜。而減少內生乳癌激酶會抑制細胞的延展也暗示了乳癌激酶在細胞移動性上的影響。最後，我們證實了乳癌激酶能夠經由p190RhoGAP的作用下增進細胞增生。綜觀以上，我們發現了乳酸激酶參與的嶄新訊息傳遞路徑以及其在生物學上可能扮演的角色，並提供了乳癌激酶與細胞移動性及細胞增生上可能的連結，這樣的連結也說明了乳癌激酶在癌症形成過程的重要性。Breast tumor kinase (Brk) is a nonreceptor tyrosine kinase that belongs to Src-like kinase family that containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other caner types. However, the molecular mechanism of that Brk participates in tumorigenesis remains to be characterized. Brk overexpression has been shown to sensitize mammary epithelial cells to epidermal growth factor (EGF). In this study, we have generated antibody against Brk which specifically recognizes both exogenous and endogenous Brk. Moreover, this Brk specific antibody can be utilized for immunoprecipitation to pull down both exogenous and endogenous Brk. In addition, we have characterized p190RhoGAP as a novel substrate of Brk and further identified the major phosphorylation site Y1105. Furthermore, we have discovered the involvement of Brk in EGF-induced p190RhoGAP phosphorylation on Y1105. This phosphorylation promotes the membrane recruitment of p190RhoGAP in response to EGF stimulation. In addition, the result that knockdown of Brk can inhibit the cell spreading implies the biological effect of Brk in cell motility. Finally, we have demonstrated that Brk is capable of promoting cell proliferation through a p190RhoGAP-dependent manner. Together, our findings identify new signaling and biological roles of Brk and indicate the potential link between Brk and both cell motility and proliferation which may contribute to its involvement in tumorigenesis.Table of Content 2
中文摘要 4
Abstract 5
Introduction 6
Signal transduction in tumorigenesis 6
Src family tyrosine kinases (SFKs) 8
Breast tumor kinase (Brk) 10
p190RhoGAP 13
Materials & Methods 17
Cell culture and transfection 17
GST fusion proteins 17
Immunization procedure 18
Antibodies 18
Immunoprecipitations 19
Western blot analysis 19
Cell spreading assay 20
Subcellular fractionation 20
Immunofluorescence and microscope 21
Cell proliferation assay 21
Results 22
Generation and characterization of the polyclonal antibody against Brk 22
Brk phosphorylates p190RhoGAP on the Tyr1105 residue 22
Brk mediates EGF-induced tyrosine phosphorylation of p190RhoGAP 23
Knockdown of Brk inhibits cell spreading 24
Brk mediates EGF-induced membrane recruitment of p190RhoGAP 25
p190RhoGAP is required for the cell proliferative effect of Brk 26
Discussion 28
Reference 34
Figures 39
Figure 1. Characterization of the anti-Brk antiserum. 39
Figure 2. Brk phosphorylates p190RhoGAP at Y1105 in vivo. 39
Figure 3. Brk mediates EGF-induced p190RhoGAP phosphorylation. 42
Figure 4. Knockdown of Brk inhibits cell spreading. 43
Figure 5. Brk enhances EGF-induced translocation to membrane fraction of p190RhoGAP. 45
Figure 6. Brk promotes membrane localization of p190RhoGAP upon EGF stimulation. 46
Figure 7. Membrane localization of p190RhoGAP induced by EGF treatment is dramatically elevated by Brk while BrkKM can dominantly inhibit the enhancement. 47
Figure 8. Brk promotes cell proliferation in a p190RhoGAP-dependent manner. 4
