https://scholars.lib.ntu.edu.tw/handle/123456789/142415
Title: | 海星卵母細胞成熟期間PRK2調節機制之探討﹝1/3﹞ | Authors: | 李士傑 | Keywords: | 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 4E;PI3 kinase;adneylyl cyclase 和Rho;starfish;oocyte maturation;PRK2;eIF4E, PI3 kinase and adenylyl cyclase | Issue Date: | 31-Jul-2002 | Publisher: | 臺北市:國立臺灣大學漁業科學研究所 | Abstract: | 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( Pisaster ochroceus),因此本試驗乃 改用澎湖地區特有之飛白楓海星( Archaster typicus)及由澳洲Tasmania 進口之海星 ( Asterias amurensis),測試其基礎卵母細胞 成熟之活性以供後續之研究。同時利用 Pisaster ochroceus之PRK2 抗體我們可由 Asterias amurensis 卵母細胞中以免疫沉澱 法萃取出PRK2 蛋白質,而飛白楓海星卵 母細胞中雖可以西方墨點法偵測到 PRK2,但其結果卻不是很恆定,現正以免 疫沉澱法測試中。 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 and Asterias amurensis from Tasmania, Australia. We have established a system for testing the responses of Archaster typicus and oocytes to maturation hormone. We also demonstrated the presence of PRK2 protein in Asterias amurensis and possibly Archaster typicus oocytes by Western blotting or immunoprecipitation against Pisaster ochraceus PRK2 antibody. 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 and Asterias amurensis oocytes are underway. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/20553 | Other Identifiers: | 902313B002320 | Rights: | 國立臺灣大學漁業科學研究所 |
Appears in Collections: | 漁業科學研究所 |
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