Melanoma differentiation-associated gene-7 protein physically associates with the double-stranded RNA-activated protein kinase PKR
Melanoma differentiation-associated gene-7 protein physically associates with the double-stranded RNA-activated protein kinase PKR
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DOI：
10.1016/j.ymthe.2005.01.018
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发表时间：
2005-05-01
影响因子：
12.4
通讯作者：
Swisher, SG
中科院分区：
文献类型：
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作者：
Pataer, A;Vorburger, SA;Swisher, SG
我们先前报道，腺病毒介导的黑色素瘤分化相关基因-7(Ad-MDA7；批准的基因符号IL24)的过表达导致PKR的快速诱导及其下游靶点的激活，从而诱导人肺癌细胞凋亡。为了探讨Ad-MDA7激活PKR的机制，我们研究了MDA-7和PKR蛋白之间的相互作用。在Ad-MDA7转导肺癌细胞后，细胞内和细胞外产生MDA-7蛋白，导致剂量和时间依赖的PKR诱导。纯化的细胞外MDA-7蛋白不能诱导PKR和细胞凋亡，提示Ad-MDA7介导的PKR激活和凋亡不依赖于细胞外的MDA-7蛋白。在Ad-MDA7转导后，RT-PCR显示尽管PKR蛋白水平增加，但PKR mRNA水平没有增加，这表明MDA-7对PKR具有转录后调控作用。免疫荧光和免疫共沉淀研究表明，MDA-7蛋白与PKR在物理上相互作用。用Ad-MDA7转导PKR+/+和PKR-/-转化的MEF后，PKR+/+细胞裂解产物中的MDA-7和PKR蛋白被磷酸化，而PKR-/-细胞中的MDA-7和PKR蛋白不被磷酸化。这些发现确定了MDA-7的第一个结合伙伴，并表明PKR和MDA-7之间的直接相互作用可能对PKR的激活和诱导细胞凋亡起重要作用，可能是通过MDA-7磷酸化或激活其他下游靶点。
We previously reported that adenoviral-mediated overexpression of the melanoma differentiation-associated gene-7 (Ad-mda7; approved gene symbol IL24) leads to the rapid induction of PKR and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. To evaluate the mechanism by which Ad-mda7 activates PKR, we studied the interaction between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, intracellular and extracellular MDA-7 protein was generated, leading to dose- and time-dependent PKR induction. Purified MDA-7 protein administered extracellularly did not induce PKR or apoptosis, suggesting that Ad-mda7-mediated PKR activation and apoptosis were not dependent on extracellular MDA-7 protein. Following Ad-mda7 transduction, RT-PCR demonstrated no increase in PKR mRNA levels despite increased levels of PKR protein, suggesting posttranscriptional regulation of PKR by MDA-7. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 protein physically interacts with PKR. Transduction of PKR+/+ and PKR-/- transformed MEFs with Ad-mda7 demonstrated phosphorylated MDA-7 and PKR proteins in the lysates of PKR+/+ but not PKR-/- cells. These findings identify the first binding partner for MDA-7 and suggest that direct interaction between PKR and MDA-7 may be important for PKR activation and apoptosis induction, possibly through MDA-7 phosphorylation or activation of other downstream targets.