The study of Growth arrest-specific 7 Involves in Cyclin-dependent kinase 5/p35 Kinase complex

細胞週期蛋白依賴型激酶5(Cdk5)及其激動分子p35主要調控老鼠腦部發育的過程。近來發現，Cdk5/p35複合體位於細胞高基氏體中，並且負責液泡運送的調控，但是該分子機制尚不清楚。生長休止基因七 (Gas7)被發現大量表現在細胞生長休止階段 (G0 phase)，並已知主要表現在腦部。CBP1是Gas7羧基(COOH)端的交互作用分子，且其序列和p35的交互作用分子，稱為C48，有高度的相似性，但是目前對於該交互分子---C48，的生物功能仍不了解。此篇論文主要是探討： Gas7是否會藉由CBP1和Cdk5及p35形成複合體，並且扮演維持高基氏體的正常結構並進一步調控其細胞功能，如液泡運送的角色。利用谷胱甘肽S-轉移(GST)的沉澱分析(GST pull down assay)，得知Gas7無法直接與Cdk5及p35交互作用，而是必須藉由CBP1。經由海馬回初代細胞的免疫染色發現，CBP1、Gas7、Cdk5和p35都有表現在細胞高基氏體。並且在老鼠腦部的發育時期，藉由蔗糖梯度法分離高基氏體(Golgi enriched sucrose gradients)，Gas7和p35會主要表現在高基氏體並和Cdk5及actin形成複合物。進一步利用定點突變蛋白VSVG-ts045-GFP蛋白運送分析，在大量表現CBP1的細胞中，會造成液泡從內質網運送到高基氏的過程有延後的現象。總結來說，Gas7及CBP1會與Cdk5及p35形成複合體，並且同樣會位於細胞高基氏體中，而CBP1似乎參與細胞液泡運送調控的機制。Cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, which is dependent on association with its neuronal specific activator, p35, is required for proper development of a mouse brain. Recently, the Cdk5-p35 kinase has been detected in the Golgi apparatus and is a factor in the regulation of membrane traffic, although the action of Cdk5-p35 in the Golgi apparatus is largely unclear. The growth arrest-specific 7 (Gas7) is identified in G0 phase and expressed preferentially in the brain. Gas7 C’-terminal Binding Protein, CBP1, is highly homologous with p35-interacting protein, but its biological function is not understood. Moreover, Gas7 is associated with actin assembly that is responsible for protein transport in Golgi apparatus. This study will determine the complex formation of Gas7, CBP1, p35 and Cdk5 tetra-complex, and their roles in Golgi architecture and Golgi-mediated cellular functions including vesicle transport. In protein-protein relationships, no direct interaction been detected between Gas7 and p35/Cdk5 kinase without mediation by CBP1. Preliminary results indicate that the pattern of Golgi distribution by analyzing specific Golgi markers of sucrose gradient prepared from Gas7 wild-type developing mouse brains reveals Gas7 and p35 are predominantly expressed in Golgi-enriched fractions. Moreover, the tetra-complex formation of Gas7, actins and Cdk5/p35, exists in Golgi-enriched microsomal fractions purified from the developing mouse brain. In the VSVG-ts045-GFP transport assay, CBP1 delays the vesicle transportation from the ER to the Golgi apparatus. Collectively, these results indicate that CBP1, a key component for Gas7 and p35/CDK5 complex formation, is not only predominantly expressed in the Golgi complex but also plays a role in protein trafficking.Table of Contents..…………………………………………………………1 Abstract….……………………...…………………………………………...3 中文摘要……….……………………………………………………………..4 Introduction………………………………………………………………...5 Materials and Methods…………………………………………………...9 Subcellular fractionation……………………………………..….9 DNA constructions………….……………………………..……10 Cell culture and Transfection.……………………….………..10 Immunostaining……………….……………………….………..11 Western blot analysis…………………………………………..12 Expression and purification of proteins from E.coli………12 GST pull-down assay…………………………………………...13 Immunoprecipitation……………………………………………14 VSVG-ts045-GFP anterograde transport assay……………15 Results…………………………………………………………………….16 CBP1 is identified as the Gas7 C’-terminal binding protein in mouse brain………………………………….……...16 Gas7 interacts with CBP1 directly in vitro…………………..17 CBP1 mediates Gas7 binding to the p25/Cdk5 kinase in vitro…..…………………………………………………………18 Gas7, Cdk5, p35 and CBP1 are localized in the Golgi apparatus in hippocampus primary culture………………...19 Gas7, p35 and Cdk5 are expressed in the Golgi apparatus in postnatal mouse brain....................................20 Gas7 is involved in p35/Cdk5 kinase in microsomal fraction in vivo…………………………………………………...21 Transport of nascent protein from the ER to the Golgi apparatus is delayed in cells expressing C’-terminal domain of CBP1 …………………..………..…………………...22 Tables and Figures………………………………………………………24 Figure 1. CBP1 protein residues are highly identical with Cdk5Rap2 and C48…………...........…………………………...24 Figure 2. Gas7 directly interacts with CBP1, but not p25, p35 and Cdk5…………………………………………………….25 Figure 3. The third coiled-coil domain is Gas7-binding region in C’-terminal of CBP1…………………………………26 Figure 4. CBP1 is a mediator connecting the interaction between Gas7 and p25 or Cdk5……………………………….27 Figure 5. Among Gas7, CBP1, p35 and Cdk5 are localized in the Golgi apparatus in the hippocampus primary culture……………………………………………………………..28 Figure 6. Gas7 and p35 are predominantly expressed in Golgi enrich sucrose gradients……………………………….29 Figure 7. Cdk5 immunoprecipitates Gas7 from a microsomal fraction.……………………………………………30 Figure 8. VSVG-ts045-GFP is delayed to transport from the ER to the Golgi apparatus in the cells ecpressing CBP1 proteins……………………………………………………31 Discussion………………………………………………………………..32 Reference…………………………………………………………………39 Appendix………………………………………………………………….4