李士傑2006-07-262018-07-062006-07-262018-07-062001-07-31http://ntur.lib.ntu.edu.tw//handle/246246/20551未成熟動物卵母細胞其減數分裂暫停 於第一次減數分裂之前期，直到被激活 後，減數分裂才能繼續進行。海星卵母細 胞受其成熟激素(1-methyladenine, 1-MA)之 刺激卵內蛋白磷酸脢C 二號相關磷酸脢 (protein kinase C-related kinase 2, PRK2)活 性會升高，繼之以成熟促進因子(maturation promoting factor, MPF)及蛋白質新生成機 制之活化。蛋白質之新生成主由真核細胞 起始因子4E (eukaryotic initiation factor 4E, eIF4E)之磷酸化來調控，最近我們更証明了 PRK2 可在活體外將eIF4E 磷酸化，且 PRK2 之活性並受到PI3 kinase 及adneylyl cyclase 之調節，因此PRK2 極可能在PI3 kinase 及adenylyl cyclase 下游來調控卵母 細胞之成熟過程。但因以上所述PRK2 相 關試驗均在活體外完成，為證明PRK2 真 是扮演我們所推測之角色，此本計劃將以 活體的試驗來測試各種PRK2 激活及抑制 劑，包括自體活化(constitutively active)及負 面優勢(dominant negative)之PRK2，對海星 卵母細胞PRK2 活性及卵母細胞成熟之影 響。因台灣地區無法取得先前試驗用之海 星品種，因此本試驗乃改用澎湖地區特有 隻飛白楓海星，並測試其基礎卵母細胞成 熟之活性以供後續之研究。目前本實驗室 已成功地將PRK2 catalytic domain 第666 個氨基酸 lysine 改變成arginine 而使之成 為dominant negative 之PRK2 並同時製備 了去除N端調節區塊之constitutively active PRK2. 其對卵母細胞成熟之影響則正在測 試當中。Animal oocytes grow and acquire all the required mRNA and proteins for the early embryonic development. After growth oocytes arrest in the prophase of meiosis I until the stimulation of maturation hormone. In starfish, maturation hormone, 1-methyladenine (1-MA), triggers the activation of maturation promoting factor, which is a major driving force for the resumption of meiosis. At the mean time, the protein synthesis machinery is also activated for translating stored mRNA. 1-MA is known to induce the activation of inhibitory G protein that leads to a bifurcate pathway including the activation of PI3 kinase and inactivation of adenylyl cyclase. Although, we know both the increase in PI3 kinase and the decrease in adenylyl cyclase activities are required for activation of MPF and protein synthesis, the downstream effectors of these signaling pathways are unclear. Shortly after the induction of 1-MA, there is an increase in protein kinase C like activity, which precedes the activation of MPF and initiation of protein synthesis. Later, this PKC-like activity is proved to be mainly due to the protein kinase C-related kinase 2 (PRK2). The initiation of protein synthesis is activated by phosphorylation of eukaryotic initiation factor 4E (eIF4E). Recently, I further demonstrated that PRK2 phosphoryaltes eIF4E in vitro. (Lee et al., 2000. Dev Biol. 228, 166-180.). In addition, PRK2 activity is regulated by 1-MA-induced PI3 kinase and adenylyl cyclase-dependent pathway. These observations suggest PRK2 is the key molecule mediating this G-protein-induced event during meiotic maturation in starfish. However, the definite proof awaits PRK2 functional study in vivo. This study aims to elucidate the role and regulation of PRK2 during meiotic maturation in starfish oocytes. Specifically, I will ask whether the role of PRK2 in vivo can be determined using known PRK2 activators and inhibitors, including a constitutively active and a dominant-negative PRK2. Due to the lack of the same starfish species used in previous studies, our laboratory is trying to use a native starfish, Archaster typicus, collected at the beach of PenHu. We have established a system for testing the responses of Archaster typicus oocytes to maturation hormone. In addition, we also generated (1) a constitutively active PRK2 by removing the regulatory domain and (2) a dominant-negative mutant by changing PRK2 amino acid 666 from a lysine to an arginine. The effects of PRK2 mutants described on the maturation responses of Archaster typicus oocytes are underway.application/pdf53725 bytesapplication/pdfzh-TW國立臺灣大學漁業科學研究所海星卵母細胞成熟蛋白磷酸 脢C 二號相關磷酸脢真核細胞起始因子 4EPI3 kinaseadneylyl cyclase 和Rhostarfishoocyte maturation, PRK2eIF4EPI3 kinase and adenylyl cyclasejournal articlehttp://ntur.lib.ntu.edu.tw/bitstream/246246/20551/1/902313B002257.pdf