標題: 利用創新的蛋白質交互作用模型研究微管上 Protein Kinase A
Studying the Function of Microtubule-associated Protein Kinase A on Neurite-like Cell Protrusion through a Novel Protein-Protein Interaction Model
作者: 曾子歡
黃兆祺
分子醫學與生物工程研究所
關鍵字: 微管;類神經細胞突起;protein kinase A;microtubule
公開日期: 2011
摘要: 神經突型態發生 (neurite morphogenesis) 是一個神經細胞生長並分化出軸突與樹突的過程,目前已知微管細胞骨架與微管相關蛋白在此過程中扮演了相當關鍵的角色。本實驗室先前的蛋白質體研究顯示,在神經分化及神經突型態發生後,protein kinase A (PKA) 在微管上的含量會顯著地提升。雖然目前已知多種細胞機制都需PKA的參與,但PKA在神經突型態發生中組織微管結構的功能目前仍不清楚。因為PKA參與了許多種細胞機制,直接抑制PKA蛋白質表現將會造成許多和神經突型態發生無關的表現型(phenotype)。為了避免此種情形,我們發展出一套能夠改變微管上PKA含量卻不影響胞內整體PKA表現量的方法。此方法讓我們得以檢驗PKA在微管上的功能。 在本研究中,我們採用了動態性蛋白質異源二聚化法 (dynamic heterodimerization method),以人工誘發特定蛋白質間的交互作用,此技術使我們得以將細胞內的PKA複合體從微管上重新分配到細胞膜。本系統由三個核心原件所組成:F2E domain、FRB domain以及AP21967 (rapamycin的衍生化合物)。在含有AP21967之環境下,F2E與FRB此兩種domain會形成緊密的異源二聚體。本研究中使用了自行建構的兩個融合蛋白質體:帶有FRB domain的PKA type II α regulatory subunit (FRB-PKA-RIIα),以及帶有F2E domain的myristoylation signaling peptide (myr-F2E)。當這些融合質體被送進細胞表現後,我們即可透過AP21967重新分配微管上的PKA到細胞膜。而我們的實驗結果顯示,當PKA被導向細胞膜時細胞傾向產生較短的細胞突,由此可得知位於微管上的PKA在類神經突細胞突起之延長過程中扮演重要的角色。
Neurite morphogenesis is a process through which neurons generate their widespread axon and dendrites. It has been shown that the microtubule cytoskeleton and microtubule-associated proteins are important for this process. Previous proteomic study from our lab indicated that the amount of protein kinase A (PKA) on microtubules significantly increased after neuronal differentiation and neurite morphogenesis. PKA has been shown to play essential roles in various cellular processes. However, its function on microtubule organization during neurite morphogenesis remains elusive. Since PKA is essential for various cellular functions, globally suppressing PKA genes will causes a plethora of phenotypes unrelated to neurite morphogenesis. To circumvent this problem, we developed a method to specifically alter the amount of PKA on microtubules without changing the overall PKA level. This allows us to determine the specific function of PKA on microtubule. In this study, a dynamic heterodimerization method was utilized. This method allows us to generate artificial protein-protein interactions, hence enables us to redistribute the PKA complex from the microtubule cytoskeleton onto the cell membrane. The system is composed of three components, the F2E domain, the FRB domain and AP21967 (an analog of rapamycin). F2E and FRB domains form a tight heterodimer in the presence of rapalog. We generated 2 fusion proteins, an FRB domain fused to the PKA regulatory subunit IIα (FRB-PKA-RIIα) and an F2E domain fused to a myristoylation signaling peptide (myr-F2E). When transfected into cells, these fusion constructs allow us to redistribute PKA from microtubules to the cell membrane upon AP2167 application. Our results indicated that the microtubule-associated PKA is important for the elongation of neurite-like cell protrusions, as cells generated shorter protrusions when PKA was redirected to the cell membrane.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079929505
http://hdl.handle.net/11536/49975
Appears in Collections:Thesis