林炎壽Yenshou Lin, Ph.D.林珈龍Chia-Lung Lin2019-09-052019-7-222019-09-052014http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060043059S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104063mTOR是一個典型的絲胺酸/蘇胺酸激脢，mTOR藉由與不同的蛋白結合而形成兩種不同的複合物，並調控不同的細胞生理如生長及代謝。 mTOR蛋白複合體1 (mTORC1) 包含 mLST8、 raptor及 mTOR，透過活化下游的蛋白 S6K1 及 4EP1調控蛋白質的合成，因此 mTORC1在調控細胞生長與增生扮演重要的角色。不同於 mTORC1，mTOR蛋白複合體2 (mTORC2) 包含 mLST8, mTOR, mSin1, PRR5 及 rictor。mTORC2 的下游蛋白目前已知道有PKCα, SGK1, 及 Akt，而 mTORC2最主要的功能與細胞骨架的排列有關，然而 mTORC2的訊息傳遞路徑至今還不是很明確。在我們過去的實驗中發現，於小鼠胚胎纖維母細胞將 rictor的基因剔除，利用 rictor抗體執行對野生型及剔除型細胞液的免疫沉澱法，配合質譜儀的分析後，發現一個蛋白與 rictor結合，並暫時命名為 RICAP。實驗的目的是探討 rictor及RICAP兩蛋白之間的關係。 首先，以免疫沉澱法證明內生性的 rictor及 RICAP有相互的結合，進一步利用重組基因蛋白的表現配合免疫沉澱法，我們確認 RICAP胺基酸 1~500的區段會與 rictor結合。在功能方面的研究，使用RNAi的方法，將 rictor及 RICAP 的蛋白質量減少時，發現導致 mTORC2下游蛋白 SGK1 S422位點的磷酸化程度下降，而不是 Akt S473與 PKCα S657，同時小鼠肌纖維母細胞分化成肌纖維或肌小管的效率大大降低，我們提出一假說即 RICAP可能在 mTORC2媒介了 SGK1的活性來調控了對肌肉細胞的分化，此為 mTORC2訊息傳遞範疇一重大突破。Mammalian target of rapamycin (mTOR) is a typical Ser/Thr kinase which forms two distinct complexes by associating with different components. The mTORC1 includes mLST8, mTOR, and raptor. It affects protein synthesis by phosphorylating its downstream substrates S6K1 and 4EP1. Therefore, it plays a major role in cell growth and is found to be deregulated in many tumors. The other one is mTORC2 which contains mLST8, mTOR, mSin1, PRR5 and rictor. It could regulate PKCα, SGK1, and Akt and affect actin polymerization and cell morphology. Nevertheless, the signal transduction pathway regarding of mTORC2 remains largely unknown. Utilizing mouse embryonic fibroblast (MEF) cells in which rictor gene was knocked out, we performed a series of experiments including immunoprecipitation and mass/spec analysis and found a novel rictor associated protein, temporarily named RICAP. The association between endogenous RICAP and endogenous rictor was further verified by using immunoprecipitation. Furthermore, RICAP aa 1~500 was associated with rictor by pull-down assay. SGK1 phosphorylation on S422 induced by insulin, but not Akt S473 and PKCα S657, is blocked both in rictor and RICAP knock down in C2C12 myoblast. This indicates that rictor and RICAP could have a common effect on SGK1, not other mTORC2 downstream molecules. Using specific bio-marker tropomyosin in immunocytochemistry, we also found that both rictor and RICAP RNAi decrease the differentiation efficacy of C2C12 myotubes. Thus, we hypothesize that RICAP might play a role in the mTORC2-mediated SGK1 activity which results in the regulation of muscle differentiation. Taken together, we explore a novel molecule mediating the mTORC2 signaling pathway.肌肉新生分化磷酸化myogensisdifferentiationphosphorylation