Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen.
Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen.
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DOI：
10.1002/j.1460-2075.1990.tb08306.x
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发表时间：
1990-06
期刊：
影响因子：
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通讯作者：
Shi Huang;Nan-Ping Wang;Ben Y. Tseng;Wen-Hwa Lee;E. H. P. Lee
中科院分区：
文献类型：
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作者：
Shi Huang;Nan-Ping Wang;Ben Y. Tseng;Wen-Hwa Lee;E. H. P. Lee
视网膜母细胞瘤易感基因（RB）编码一个110 kd的磷蛋白（pp 110 RB），与SV 40 T抗原和其他几种DNA肿瘤病毒的转化蛋白形成特异性复合物。与RB的相互作用被认为有助于这些病毒的转化，如遗传分析所示。为了帮助理解这些相互作用的功能，RB与T结合的区域已经被绘制出来。已经开发了能够产生全长RB蛋白的体外蛋白合成系统，以促进作图研究。当RB mRNA的5′非编码区被β珠蛋白mRNA或植物病毒RNA苜蓿花叶病毒（AMV）RNA 4的5′非编码区取代时，网织红细胞裂解物的翻译效率增加了5 - 10倍。测定从该系统产生的一系列突变RB多肽的T结合。RB蛋白的两个非连续区域，氨基酸残基394 - 571和649 - 773，被发现是与T结合所必需的：在任一区域的突变消除了T-RB复合物的形成。这些结果与以下发现一致，即在迄今为止分析的所有情况中，人肿瘤细胞中的突变RB蛋白也由于包括两个所需区域中的至少一个的缺失而不能与T抗原结合。因此，在体外定义为与T相互作用所必需的RB区域可能也是生理相关的，并且可能在正常的RB蛋白功能中发挥重要作用。
The retinoblastoma susceptibility gene (RB) encodes a phosphoprotein of 110 kd (pp110RB) that forms specific complexes with SV40 T antigen and the transforming proteins of several other DNA tumor viruses. Interaction with RB is thought to contribute to transformation by these viruses as demonstrated by genetic analyses. To help understand the function of these interactions, the regions of RB that are involved in binding to T have been mapped. An in vitro protein synthesis system capable of producing full‐length RB protein has been developed to facilitate the mapping study. A 5‐ to 10‐fold increase in translational efficiency in the reticulocyte lysate was obtained when the 5′ non‐coding region of RB mRNA was replaced with that of beta‐globin mRNA or a plant viral RNA, alfalfa mosaic virus (AMV) RNA4. A series of mutated RB polypeptides produced from this system were assayed for T binding. Two non‐contiguous regions of the RB protein, amino acid residues 394‐571 and 649‐773, were found to be necessary for binding to T: mutations in either region abolished T‐RB complex formation. These results are consistent with the finding that, in all the cases analyzed so far, mutated RB proteins in human tumor cells also failed to bind to T antigen due to deletions including at least one of the two required regions. Thus the regions of RB defined in vitro as necessary for interaction with T might be physiologically relevant as well, and might play a fundamental role in normal RB protein function.