行政院國家科學委員會專題研究計畫期中進度報告:Gfi-1B經phorbol ester 誘發降解之機制探討(1/3)

Gfi-1B （growth factor independence 1B ）為細胞原致癌基因，其所譯碼的蛋白質之胺基端 有SNAG repressor domain， 基端有 zinc finger domain，可和以AATC 為core motif 特定的 DNA 序列結合，執行其轉錄抑制子的功能。Gfi-1B 的表現具有組織特異性，主要表現於骨髓 及脾臟。在T 細胞中表現過量的Gfi-1B, 會嚴重影響到T 細胞的正常發育; 目前在小鼠實驗 中,發現Gfi-1B 基因剔除鼠, 會因為無法形成正常成熟的紅血球而死亡◦ 顯示Gfi-1B 基因對於 血液細胞,尤其是紅血球細胞的發育調控扮演著重要且具決定性的角色。在慢性髓細胞性白血 病（chronic myelocytic leukemia，CML）細胞株K562 中可見Gfi-1B 的大量表現◦ 但目前對其 在K562 細胞中的生物功能尚不清楚。我們過去的研究發現Gfi-1B 可以抑制CMV 啟動子的 活性, 在PMA 誘發K562 細胞走向分化的過程中，Gfi-1B 蛋白質的表現量會有明顯的下降趨 勢，而實驗結果顯示，PMA 是藉由ubiquitin-dependent proteasomal degradation 之作用機制， 造成Gfi-1B 蛋白質表現量的下降。PMA 調控的Gfi-1B 降解作用具有細胞特異性，可發生在 K562 細胞，而不發生在HeLa 細胞中。在Gfi-1B 蛋白質第87~103 胺基酸序列為可能的PEST region，是PMA 促使Gfi-1B 迅速降解的作用位置;並由免疫沉澱的實驗我們也發現可能是經 由SCF-mediated proteolysis 將Gfi-1B 蛋白質降解。為了對Gfi-1B 蛋白質的降解作用機制更進 一步的了解,在過去一年間，我們另外證實以下幾個事實: 一在PMA 誘發K562 細胞走向分化的過程中，除了Gfi-1B 蛋白質的表現量會有明顯的下降 趨勢之外，Gfi-1B RNA 的表現量也會有快速的下降的情形，而且Gfi-1B RNA 的表現量會受 到MAPK/ERK pathway 的調控◦ 二透過人類in vivo 磷酸化的實驗，我們發現在K562 細胞中，PMA 所誘發Gfi-1B 蛋白質 的hyperphosphorylation 的位置在PEST 序列上，而且這樣的hyperphosphorylation 現象並不會 出現在HeLa 細胞中◦ 三Gfi-1B 蛋白質上第46-164 個胺基酸序列是proliferating state 調控Gfi-1B 蛋白質降解的作 用位置◦Gfi-1B （growth factor independence 1B ） is a cellular proto-oncogene that encodes a transcriptional repressor with an N-terminal SNAG repressor domain and a C-terminal zinc finger domain. Gfi-1Bexpression is restricted to bone marrow and spleen. They can bind to a sequence containing of an AA(T/G)C core. Overexpression of Gfi-1B in T cells leads to defects in T cell lineage commitment. Targeted gene disruption of Gfi-1B in mice results in embryonic lethality due to a profound defect in formation of enucleated erythrocytes, suggesting that an essential role of Gfi-1B in blood cell development, specifically within the erythroid and megakaryocytic cell lineages. Gfi-1B was found to express at very high levels in the CML (chronic myelocytic leukemia) line K562. Its biological function in K562 cells is still unclear. The purpose of this study is to investigate the regulation of Gfi-1B expression in K562 cells. Our prelimary data have showed that Gfi-1B could act as a transcription repressor for the CMV promoter, and found that Gfi-1B is specifically degraded in PMA-treated K562 cells but not in HeLa cells. We further demonstrated that the degradation of Gfi-1B is through ubiquitin-proteasome system. By sequence analysis, all Gfi-1B coding sequences among different species contain a PEST-like sequence, which is considered as a potential protein degradation signal, which deletion stabilized its protein expression. In addition, the hGfi-1B was found to be associated with Skp1, which is a component of SCF complex ( Skp1-Cullin-1/CDC53- F-box/Roc), in PMA-treated K562 cells. We proposed that PMA-induced degradation of hGfi-1B is mediated through the SCF pathway via a PEST sequence. We have analyzed the detailed molecular mechanism of Gfi-1B degradation. Here are our recent findings: 1. Inhibition of MEK-mediated signal prevents PMA-induced instability of Gfi-1B RNA transcripts. 2. PMA induces the rapidly degraded and hyperphosphorylation through PEST sequence of Gfi-1B protein in K562 cells but not in PMA-treated HeLa cells. 3. The region of amino acid 45 to 164 of Gfi1B is also required for proteosomal degradation in proliferating state