陳瑞華臺灣大學：分子醫學研究所陳忻怡Chen, Hsin-YiHsin-YiChen2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51385乳癌激酶（Breast tumor kinase, Brk）是1995年自轉移性乳癌腫瘤中所找到的非受體型酪胺酸激酶（non-receptor tyrosine kinase）。由於其氨基酸序列（amino acid sequence）及蛋白質區塊分析（domain structure analysis）都和Src有很高的相似性，因此被列為src酪胺酸激酶家族中的一員。先前的文獻指出，在三分之二的乳癌腫瘤、黑色素細胞瘤以及大腸癌中都可以發現乳癌激酶被大量的表現。這暗示著乳癌激酶極有可能在細胞致癌機轉（oncogenesis）中扮演某種重要的角色。然而，現今的研究對於乳癌激酶的生物功能、調控方式及其受質（substrate）的瞭解仍十分有限。因此，在本篇論文中，我們找到了兩個乳癌激酶的受質以及其所參與的訊息傳遞路徑（signaling pathway），並進而探討乳癌激酶在癌症發生（tumorigenesis）過程中所扮演的角色為何。在論文的第一部份，我們發現表皮生長因子（EGF）會活化乳癌激酶，進而磷酸化paxillin在31和118這兩個位置。這樣的磷酸化會提高CrkII和paxillin的親和力（binding affinity），使得Dock180和Elmo有辦法和已磷酸化的paxillin結合，進而促進Rac1的活性。透過這條訊息傳遞路徑，乳癌激酶可以增加細胞移動以及侵入其他組織（invasion）的能力，並參與傳遞表皮生長因子造成細胞移動能力增加的這條訊息傳遞路徑。在論文的第二部份，我們找到了乳癌激酶的另一個受質─p190RhoGAP。乳癌激酶會磷酸化p190RhoGAP在酪胺酸1105的位置，繼而增強p190RhoGAP以及p120RasGAP的結合能力。當這兩個蛋白質結合之後，一方面會增加p190RhoGAP抑制RhoA活性的能力，進而抑制RhoA的活化；另一方面會抑制p120RasGAP的活性，進而造成Ras的活化。透過這個雙向調控機制，我們發現乳癌激酶確實可以參與表皮生長因子所導致RhoA活性下降的這條訊息傳遞路徑。同時，我們也發現乳癌激酶的確可以抑制壓力纖維（stress fiber）的產生，這個現象和我們所觀察到RhoA的活性下降所造成的生物功能是一致的。此外，我們還證明了p190RhoGAP的存在對乳癌激酶在細胞移動上的調控是必須的。因此，本篇論文揭露了乳癌激酶尚未被人發現的生物功能（biological function），也提供了洞察癌化分子機轉的方式。希望透過這個研究，能提供現今目標導向的癌症治療的一個新的思考方向。Brk (Breast tumor kinase), a nonreceptor tyrosine kinase, was originally identified from a functional screen for identifying the tyrosine kinases overexpressed in metastatic breast cancers. Previous studies have shown that Brk is overexpressed in two-thirds of breast tumors and several other cancer types. However, the molecular mechanism in which this kinase participates with respect to tumorigenesis remains poorly characterized. In this thesis, we identified two substrates of Brk and raveled their signaling pathways to dissect Brk’s role in tumor progression. In the first part of study, we demonstrate that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events provide the binding site for CrkII which recruits DOCK180-Elmo to promote the activation of small GTPase Rac1. Through this pathway, Brk possesses the ability to promote cell migration and invasion and functions as a mediator of EGF-induced migratory occurrence. In the second part of this thesis, we identified that p190RhoGAP is another substrate of Brk. Brk phosphorylates p190RhoGAP at the Y1105 residue, thereby promoting p190RhoGAP association with p120RasGAP. This complex formation between p190RhoGAP and p120RasGAP stimulates the ability of p190RhoGAP to downregulate RhoA, while inhibits p120RasGAP activity toward Ras inactivation. As a result, Brk oppositely regulates RhoA and Ras and mediates EGF-induced RhoA inactivation. Consistent with its inactivation of RhoA, Brk inhibits stress fiber formation. Furthermore, we demonstrated that p190RhoGAP is required for Brk-induced chemotactic migration towards EGF. Together, our findings reveal previously unknown function of Brk in regulating three small GTPases─Rho, Rac, Ras via phosphorylating paxillin and p190RhoGAP. The identification of these signaling pathways transduced by Brk would shed light on the role of Brk in tumor progression.Table of Contents ........................................ 2 中文摘要.................................................. 5 Abstract.................................................. 6 Literature Review ........................................ 7 Tumor Progression........................................ 7 1.1 Proliferation and tumor growth ....................... 7 1.2 Migration and Metastasis ............................ 8 2. Proliferation.......................................... 9 2.1 The Phenomena of Cell Proliferation ................ 9 2.2 The Signaling Network of Cell Proliferation .......... 9 3. Migration ............................................ 13 3.1 The Phenomena of Cell Migration ..................... 13 3.2 The Concept of Migration : The Migration Cycle....... 13 3.3 Involved Signalings in Cell Migration ............... 14 4. Protein Tyrosine Kinases ............................. 21 4.1 Transmembrane Receptor Tyrosine Kinase (RTK)......... 21 4.2 Nonreceptor Tyrosine Kinase (NPTK) .................. 22 5. Small GTPases......................................... 23 5.1 GEFs & GAPs: Critical Controllers of Small GTPase ... 24 5.2 The Ras Subfamily ....................................28 5.3 The Rho Subfamily.................................... 30 6. Brk .................................................. 32 6.1 Identification and genome structure of Brk........... 32 6.2 Domain structure of Brk ............................. 32 6.3 Expression pattern of Brk.............................33 6.4 Biological role of Brk............................... 34 6.5 Identified substrates of Brk .........................35 Chapter I: .............................................. 37 Brk actives Rac1 and Promotes Cell Migration and Invasion by Phosphorylating Paxillin .................................. 37 Abstract................................................. 38 Introduction ............................................ 39 Materials and Methods ................................... 42 Cell culture, transfection, and retroviral infection..... 42 cDNA cloning and plasmid constructions .................. 42 Antibodies and reagents.................................. 43 GST fusion proteins...................................... 43 Production of baculovirus................................ 43 Immunoprecipitations..................................... 44 In vitro kinase assay ................................... 44 Assay for GTP-bound Rac.................................. 44 Immunofluorescence and confocal studies.................. 45 Cell migration assays.................................... 45 Cell invasion assays..................................... 46 Reduction of endogenous Brk expression with siRNA ....... 46 Results ................................................ 47 Brk induces paxillin phosphorylation at Y31 and Y118................................. 47 Brk interacts with paxillin in vivo .................................................................. 48 Paxillin is a direct substrate of Brk.................... 49 Brk promotes Rac1 activation ........................... 49 Brk is activated by EGF signaling....................... 50 Brk mediates EGF-induced paxillin phosphorylation and Rac1 activation ....................................... 50 Brk is colocalized with paxillin at the leading cell edges of migratory cells ..................................... 51 Brk promotes cell migration and invasion and mediates a EGF-induced migratory event.................................. 52 Discussion............................................... 55 Chapter II:................................. 59 Brk oppositely regulates RhoA and Ras by phosphorylating p190RhoGAP............................................. 59 Abstract..................................... 60 Introduction ........................................... 61 Materials and Methods ........................................................................................... 64 Plasmids.......................................................................................................... 64 Cell culture and transfections ......................................................................... 64 Antibodies and reagents.................................................................................. 64 Retroviral and lentiviral infections................................................................. 65 Immunofluoresence ........................................................................................ 65 Immunoprecipitations..................................................................................... 66 Rho and Ras activity assays ........................................................................... 66 Migration assays............................................................................................. 66 Results .................................................................................................................... 68 Brk inhibits serum and Ca2+-induced stress fiber formation .......................... 68 Brk down regulates RhoA activity ................................................................. 68 Brk knockdown inhibits EGF-induced RhoA inactivation............................. 69 p190RhoGAP is a putative substrate of Brk................................................... 69 Brk directly phosphorylates p190RhoGAP at Tyrosine 1105 in vitro ............ 70 Brk increases p120RasGAP binding to p190RhoGAP................................... 70 RhoA downregulation by p190RhoGAP is enhanced by Brk ........................ 71 Brk promotes Ras activity through p190 phosphorylation............................. 71 p190RhoGAP is required for the migratory function of Brk.......................... 71 Discussion............................................................................................................... 73 Reference ................................................................................ 76 Figure .................................................................................... 100 Appendix............................................................................... 1296149533 bytesapplication/pdfen-US乳癌激酶磷酸化受質細胞移動BrkTyrosine phosphorylationPaxillinp190RhoGAPMigrationInvasion[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51385/1/ntu-96-D91448005-1.pdf