藉由GSK3ß磷酸化Bcl2L12，調控腦瘤細胞株細胞凋亡

GSK3ß-mediated Bcl2L12 phosphorylation modulates apoptosis through caspases-3/7 in brain cancer cell.

10.6832/KMU.2010.00015

陳暐婕

肝醣合成酶激酶-3β ； 細胞凋亡 ； GSK3ß ； apoptosis

高雄醫學大學運動醫學系碩士在職專班學位論文

2019年

碩士

洪義人

繁體中文

肝醣合成酶激酶-3β (GSK3β)，屬於Serine/Threonine kinase並可調控蛋白質生成、細胞生長、分化、凋亡及發炎反應。本實驗室以GSK3β為釣餌，利用酵母菌雜交系統進行大規模篩選，找到Bcl2L12。Bcl2-Like 12(Bcl2L12)含高度保留的BH2 domain，屬於Bcl-2 family成員之一，有Bcl2L12和Bcl2L12A兩種isoform。本篇研究中，為了更證實GSK3β和Bcl2L12之間的交互作用，利用酵母菌雜交系統和Far western blot進行研究。結果顯示出Bcl2L12是藉由中間片段(153-191 a.a)和GSK3β進行交互作用，此Bcl2L12片段位於BH2 domain 外。許多文獻指出GSK3β會去磷酸化其結合蛋白，我們利用In vitro kinase assay 證實 Bcl2L12是GSK3β的受質，且磷酸化位置在S156，另一個 isoform Bcl2L12A並不是GSK3β的受質。接著要探討Bcl2L12磷酸化對於細胞凋亡的影響，分別將Bcl2L12 wild type 、Bcl2L12 mutant (S156A) 轉染到 Glioma cell lines (U87MG)。在STS處理下刺激細胞凋亡，當細胞表現Bcl2L12時，可產生抗細胞凋亡。另一方面，將Bcl2L12突變後，細胞則失去抗凋亡的功能 。更進一步，結合STS和LiCl(GSK3β抑制劑)，當細胞表現Bcl2L12時，則無法抗細胞凋亡。由以上實驗得知GSK3β會磷酸化Bcl2L12，並且調控細胞凋亡。

Glycogen synthase kinase 3 (GSK3β) is a multifunctional serine/threonine kinase and involved in multiple cellular processes including cell proliferation, differentiation, microtubule dynamic, cell cycle and apoptosis. By using large scale yeast 2-hybrid screening, Bcl2L12 was found as a GSK3ß binding partner in testis cDNA library. BCL2L12 (Bcl2-like-12) is an identified member of Bcl2 family and contain a highly conserved BH2 domain, BH3-like domain and several proline-rich. It has two splicing variants: the classical form of the gene and a truncated form called BCL2L12A. Our data showed that Bcl2L12 middle fragment (153-191 residues) which locates outside of Bcl2L12 C-terminal BH2 motif is responsible for the binding to GSK3b and also confirmed by far-western blotting experiment. No interaction was detected between Bcl2L12A and GSK3b. In vitro kinase assay showed that GSK3b phosphorylates Bcl2L12 at S156 but not Bcl2L12A (Bcl2L12 splicing transcript variant), Phosphatase were used to confirm that GSK3ß indeed phosphorylates Bcl2L12 at S156. In addition, Bcl2l12 directly decrease activation of GSK3ß Tau phosphorylation. We transfected Bcl2L12, Bcl2L12 S156A and Bcl2L12A into Glioma cell line (U87MG) and treatment staurosporine (apoptosis stimulation). The data showed that Bcl2L12 and Bcl2L12A may function as anti-apoptosis in response to staurosporine (STS) treatment. The data also demonstrated that Bcl2L12 S156A lose function. Furthermore, Bcl2L12-expressing U87MG undergo apoptosis in response to STS and LiCl (GSK3ß inhibitor) treatment. In this study we found that GSK3ß mediate apoptosis through phosphorylate Bcl2L12 at S156.