Characterization of a leucine zipper-containing protein identified by retroviral insertion in avian neuroretina cells
Characterization of a leucine zipper-containing protein identified by retroviral insertion in avian neuroretina cells
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DOI：
10.1074/jbc.271.48.30790
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发表时间：
1996-11-29
影响因子：
4.8
通讯作者：
Marx, M
中科院分区：
文献类型：
--
作者：
Proux, V;Provot, S;Marx, M
我们以前报道过，在体外用不携带癌基因的逆转录病毒RAV-1感染鸡胚神经视网膜（NR）细胞后，NR细胞被诱导增殖。NR细胞增殖是由两个相关的丝氨酸/苏氨酸蛋白激酶c-mil/c-raf或c-Rmil/B-raf基因的频繁激活和随后的逆转录病毒转导引起的。我们还发现，这些原癌基因激活的一个非常早期的事件是含有病毒和细胞序列的嵌合mRNA的合成，所述嵌合mRNA通过剪接机制连接。在当前的研究中，我们已经检测了RAV-1诱导鹌鹑NR细胞增殖的能力。通过使用逆转录聚合酶链反应技术，我们在用RAV-1感染的几种增殖的鹌鹑NR培养物中鉴定出，含有与RAV-1剪接供体位点连接的细胞序列的嵌合mRNA，这些细胞序列来源于命名为RIG的基因，其通过在几种胚胎组织中检测到的1.9 kb mRNA表达，2.3 kb的第二转录物在NR中特异性表达，其中两种转录物都受到发育调节，R10 cDNA编码一个含有亮氨酸拉链基序的251个氨基酸的多肽，它与推定的D52/N8 L蛋白具有显著的相似性，该蛋白由先前报道的在人乳腺癌和肺癌中过表达的mRNA编码，通过使用其氨基末端和含亮氨酸拉链区域的特异性多克隆抗体，我们在培养的禽类成纤维细胞中鉴定了R10基因产物为23 kDa的细胞质蛋白，在表达2，3-kb转录物的有丝分裂后NR细胞中检测到第二种30 kDa的蛋白质。我们还通过体外转录/翻译和免疫沉淀表明，R10蛋白可以很容易地形成同二聚体，推测是通过其亮氨酸拉链基序。
We reported previously that post-mitotic chicken embryonic neuroretina (NR) cells are induced to proliferate following in vitro infection with RAV-1, a retrovirus that does not carry an oncogene, NR cell multiplication results from the frequent activation and subsequent retroviral transduction of two related serine/threonine protein kinases, the c-mil/c-raf or c-Rmil/B-raf genes. We also showed that a very early event in the activation of these proto-oncogenes is the synthesis of chimeric mRNAs containing viral and cellular sequences joined by a splicing mechanism, In the current study, we have examined the ability of RAV-1 to induce proliferation of quail NR cells, By using the reverse transcription polymerase chain reaction technique, we identified, in several proliferating quail NR cultures infected with RAV-1, a chimeric mRNA containing cellular sequences joined to the RAV-1 splice donor site, These cellular sequences are derived from a gene designated RIG, which is expressed through a 1.9-kilobase (kb) mRNA detected in several embryonic tissues, A second transcript of 2.3 kb is specifically expressed in the NR, where both transcripts are developmentally regulated, The R10 cDNA encodes a 251-amino acid polypeptide that contains a leucine zipper motif, It exhibits significant similarity with the putative D52/N8L protein, encoded by an mRNA reported previously to be overexpressed in human breast and lung carcinomas, By using polyclonal antibodies specific for its amino-terminal and leucine zipper-containing regions, we identified the R10 gene product as a cytoplasmic protein of 23 kDa in cultured avian fibroblasts, A second protein of 30 kDa is detected in post-mitotic NR cells that express the 2,3-kb transcript, We also show, by in vitro transcription/translation and immunoprecipitation, that the R10 protein can readily form homodimers, presumably through its leucine zipper motif.