The Characterisic Changes in Phosphorylation and mRNA Level of Nucleophosmin/B23 during the Entry of Cells into Mitosis

• Main Authors: Jiang, Pei-Shin, 江佩馨
• Other Authors: Benjamin Yat-Ming Yung
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/49392598884828675213

碩士 === 長庚醫學暨工程學院 === 基礎醫學研究所 === 85 === Nucleophosmin/B23 是一個由 294 個氨基酸所組成的核仁磷酸 蛋白，其分子量約為 37 kDa，等電點 (isoelectric point) 約為 5.1。 由於其在細胞中多以磷酸化的形式存在，因此其功能的調節被推測可能與 其磷酸化有關。本研究的目的是想藉由 B23 的表現及其磷酸化的變化， 進一步探討 Nucleophosmin/B23 在細胞進入有絲分裂時所扮演的角色。 以 Nocodazole 處理細胞，將細胞周期停頓於有絲分裂前期 (Prometaphase)，並以雙向電泳 (2D electrophoresis) 及西方墨點法 (Western blot) 來分析經過不同時間處理的有絲分裂前期細胞中的 Nucleophosmin/B23，我們發現其有不同的形式存在；依其分子量由小至 大及等電點由鹼至酸，將其定義為形式一、二、三及四。且發現細胞由間 期 (G2) 進入有絲分裂期(M phase) 時，其磷酸化程度增加且磷酸化位置 不同;這可能意謂著此時 Nucleophosmin/B23 的磷酸化是由連續性的激活 酵素 (Kinase) 作用所完成的。此外，由北方墨點法分析 (Northern blot analysis) 得知，細胞在間期末期 (late G2) 時 Nucleophosmin/ B23 的 mRNA 的表現量較有絲分裂期時低。當將帶有 Nucleophosmin/B23 sense 及 antisense gene 的質體送入細胞表現，則會延遲細胞進入有絲 分裂。將 Flag-tagged 正常及含有缺失片段的 nucleophosmin/B23 cDNA 質體轉染於細胞中表現，在螢光顯微鏡下以 anti-flag 抗體偵測其在細 胞中的位置，發現帶有氨基酸缺失片段 #83-152 的 flag-RN 無法進入核 仁，而 flag-Alu (Δ117-186) 則可進入;這顯示氨基酸片段 #83-117 可 能含有 nucleolus translocation signal (NoLS)。 Nucleophosmin/B23, a nucleolar phosphoprotein, is consisted of 294 amino acids. Its molecular weight is about 37 kDa and the isoelectric point (pI) is 5.1. The phosphorylation of nucleophosmin/B23 has been revealed, and it may regulate the function of nucleophosmin/B23. The mitotic form of nucleophosmin/B23 has been defined and revealed highly phosphorylated during mitosis. By two-dimensional electrophoresis, the multiple forms of nucleophosmin/B23 is first demonstrated and defined as form 1, 2, 3 and 4 beginning with the least acidic and fastest migrating spot during the nocodazole-arrested mitosis. Moreover, the increasing level and different sites of phosphorylation may implicate that the cascade of continuous phosphorylation existed at G2/M transition. It was found that mRNA level of nucleophosmin/B23 was the highest at M phase. Transfection of B23 antisense plasmid inhibited nucleophosmin/B23 expression and delayed the G2/M progression. It indicated that nucleophosmin/B23 played a functional role at G2/M transition. By transfection of flag- tagged nucleophosmin/B23 construct deleted at different phosphorylation sites, the phosphorylation may not correlate with G2/M progression. It was found that flag-RN (Δ83-152) truncated protein could not enter into nucleolus at interphase, but flag-Alu (Δ 117-186) could. It may implicate that there was nucleolus translocation signal (NoLS) in amino acid #83-117.