Arl6ip1與Ran在中心體之交互作用參與細胞週期的過程

Abstract

ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) 是一個多功能的蛋白質，它調控glutamate的傳遞、細胞分化及細胞凋亡的過程，然而 Arl6ip1 這樣多功能的蛋白質是否有其他蛋白質與其結合進而參與許多不同功能的調控目前仍不清楚。因此，首先我們利用 anti-Arl6ip1 antibody-conjugated protein G beads 與 3 hours post-fertilization (hpf) 的斑馬魚胚胎之萃取總蛋白進行 immunoprecipitation assay (IP) 偵測 Arl6ip1 的結合蛋白。經 LC-MS/MS 分析 IP 下來的蛋白中，有一個屬於 Ras superfamily 稱為 Ras-related nuclear protein (Ran) 的 GTPase 蛋白最有可能與 Arl6ip1 結合。為了確認 Arl6ip1 會與 Ran 結合，我們再用 mouse anti-Ran antibody 進行 Western blotting 分析 IP 後的產物，發現 Ran 的確存在。另外，以 Escherichia coli 表現 Arl6ip1 和 Ran 的重組蛋白，進行 IP 後經由 Western blotting 分析也能得到 Ran positive 的相同結果。為了進一步了解哺乳動物中 Arl6ip1 與 Ran 是否同樣具有交互作用，我們以人類細胞株 HEK 293T 進行 IP 實驗，結果也發現 Ran 會藉由 IP 被 anti-Arl6ip1 antibody-conjugated protein G beads 所抓下來，故證實無論在斑馬魚或是哺乳動物細胞， Ran 與 Arl6ip1 皆具有交互作用。接著我們利用 whole mount in-situ hybridization 觀察 arl6ip1 mRNA 與 ran mRNA 在斑馬魚胚胎中各時期的表現，發現在斑馬魚胚胎發育前期 ran mRNA 表現於整個胚胎，到了 30 hpf 則表現於軀幹、頭部及眼睛區域，而在 48 hpf 時， ran mRNA 的表現慢慢局限至頭部和眼睛，此與 arl6ip1 mRNA 表現型態類似，顯示 ran mRNA 與 arl6ip1 mRNA 在斑馬魚胚胎發育的不同時期中會共同表現於特定組織。更進一步的，我們想觀察 Arl6ip1 及 Ran 在細胞中結合的位置，於是利用 Arl6ip1 及 Ran 抗體以 HEK 293T 進行細胞免疫染色，發現 Arl6ip1 與 Ran 在整個細胞週期中皆會共同表現於中心體 (又稱為微管組織中心)。綜合以上結果，證明 Arl6ip1 在整個細胞週期的過程中皆會與Ran共同結合在中心體，而 Arl6ip1 與 Ran 的結合作用可能參與細胞分裂時紡錘體的形成或是調控細胞內微管的組成。

ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a multi-function protein and it is involved in regulating glutamate transport, differentiation and apoptosis. However, whether any protein associated with Arl6ip1 to mediate different function remains unknown. We performed immunoprecipitation (IP) assay, in which the total proteins extracted from zebrafish embryos were mixed with anti-Arl6ip1 antibody-conjugated protein G beads, and analyzed by LC-MS/MS of this IP product. Results showed that Ras-related nuclear protein (Ran), a GTPase protein of Ras superfamily, was the most putative protein that may interact with Arl6ip1. Using mouse anti-Ran antibodies to perform Western blot analysis of IP product, we found that Ran was detected. In addition, when recombinant Arl6ip1 and Ran produced by Escherichia coli was used to carry out IP, Ran was also positively detected. In order to know whether Arl6ip1 interacts with Ran in mammals, we prepared HEK 293T cell extracts and performed IP, resulting that Ran was also pull down by anti-Arl6ip1 antibody-conjugated protein G beads. Therefore, we demonstrated that Arl6ip1 could interact with Ran not only in zebrafish embryos but also in mammalian cell line. Furthermore, using whole-mount in situ hybridization, we observed that the dynamical expression patterns of ran mRNA were co-localized with those of arl6ip1 mRNA at various developmental stages. To study the subcellular localization of these protein, we performed immunocytochemistrical experiments by using antibodies against Arl6ip1 and Ran in HEK 293T cells, we found that both Arl6ip1 and Ran signals were co-localized at centrosome, which is the main microtubule organizing center, throughout cell cycle progression. Taken together, we demonstrated that the Arl6ip1 interacts directly with Ran through the cell cycle at centrosome. This novel protein-protein interaction may play a role in controlling the spindle checkpoint during mitosis and the regulation of microtubule organization.