Summary: | 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.
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