選殖及鑑定一含 d4－domain 之基因

Abstract

老鼠 Requiem 基因為 IL－3 存活依賴性鼠類骨骷細胞株 FDCP-1 缺乏生存因數 IL－3 時誘導 FDCP-l 細胞株進入細胞自戕程式所必須之蛋白，屬於 d4－family 的一員。為找尋 d4－family其他成員，利用 d4－domain 比對 Genebank ，同時經由分子選殖得到一 cDNA , R5。利用北方墨點分析法可得到 R5 mRNA 在不同組織及細胞株的表現及分佈情形。以 R5 DNA 序列比對可得到小鼠與線蟲之 R5 homologs 。以 R5 之S31B 區域（胺基酸 102－288）所製作的多源抗體可免疫共沉澱 K562 細胞內之 R5 蛋白質與 R5－d4- cDNA 表現之蛋白質。以 d4－domain 表現出來的重組蛋白質（胺基酸 289 －498 ）可與 HeLa 細胞內的數種蛋白質產生交互作用，證實 d4－ domain 為 R5 與其他蛋白質的交互作用區域。 R5具有多個 DNA－dependent protein kinase ( DNA－PK ）磷酸化位置，經 DNA - PK 激?分析法證實為其良好的 in vitro 受質。 R5 可受 DNA－PK 的磷酸化推測可能在 DNA－PK 多重調節功能上扮演重要的角色。

Requiem, a mouse gene activated during apoptosis induced by IL-3 deprivation of the IL-3-dependent murine myeloid cell line FDCP-1, belongs to members of d4-family with a d4-domain in its C-terminus. A novel d4-family gene, human R5 was cloned based on the homology to requiem d4-domain. Mouse and C.elegans R5 homologs were also identified by blasting Genebank database with human R5 sequence. Northern blots were performed with mRNA from various human tissues and cell lines. Polyclonal antibody against S31B region (aa.102-288) could be used for immunoprecipitating R5 from K562 cells and in vitro transcription/translation product of R5-d4+ cDNA. Recombinant protein R5-S31d4-GST (aa.289-498) containing d4- domain was found to interact with various proteins in HeLa cells. There are at least 5 putative DNA-PK phosphorylation sites in R5 sequence and were identified. The fact that R5 protein can indeed be phosphorylated by DNA-PK in vitro suggests that DNA-PK may serve as a potential regulator of R5 in vivo.