Ma Y, Geerdes D W, Vogt P K
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Oncogene. 2000 Oct 5;19(42):4815-21. doi: 10.1038/sj.onc.1203834.
Some functions of the Qin oncoprotein are not dependent on DNA binding. In order to test the requirement for DNA binding in Qin-induced oncogenic transformation, site directed mutations were introduced in the winged helix (WH) DNA binding domain of the Qin protein. In cellular Qin (c-Qin), the glycine at position 233 was either deleted or substituted with the amino acids aspartic acid, alanine, glutamic acid, asparagine, proline or lysine. The same position carries aspartic acid in the viral Qin protein (v-Qin). The adjacent residues, threonine 232 and lysine 234, were separately mutated to proline. Several additional amino acid substitutions believed to be involved in DNA contacts were introduced at the following c-Qin positions: asparagine 189, histidine 193, serine 196 or arginine 236. Most of the substitutions reduced DNA binding of Qin, one mutation, H193A, completely abolished DNA binding, and another mutation, T232P, increased DNA binding affinity. Mutant H193A failed to transform chicken embryo fibroblasts (CEF), all other mutants, even those showing minimal DNA binding, retained oncogenicity for CEF. The efficiencies of focus formation induced by these mutant proteins in cell culture were not significantly different from that of wild type. However, the rate of focus development and the size of foci induced by the Qin mutants were greater with strong DNA binders than with weak DNA binders. Transdominant negative constructs consisting of the winged helix domain of cQin or v-Qin interfered with focus formation induced by full length Qin proteins. These results suggest that DNA binding is a prerequisite for transformation by Qin, and strong DNA binding is related to accelerated transformation in CEF.
秦癌蛋白的一些功能不依赖于DNA结合。为了测试秦诱导的致癌转化中DNA结合的必要性，在秦蛋白的翼状螺旋（WH）DNA结合结构域引入了定点突变。在细胞秦蛋白（c-Qin）中，第233位的甘氨酸被删除或被天冬氨酸、丙氨酸、谷氨酸、天冬酰胺、脯氨酸或赖氨酸取代。病毒秦蛋白（v-Qin）的相同位置是天冬氨酸。相邻残基苏氨酸232和赖氨酸234分别突变为脯氨酸。在以下c-Qin位置还引入了几个据信参与DNA接触的额外氨基酸取代：天冬酰胺189、组氨酸193、丝氨酸196或精氨酸236。大多数取代降低了秦蛋白与DNA的结合，一个突变体H193A完全消除了DNA结合，另一个突变体T232P增加了DNA结合亲和力。突变体H193A未能转化鸡胚成纤维细胞（CEF），所有其他突变体，即使是那些显示出最小DNA结合的突变体，对CEF仍保留致癌性。这些突变蛋白在细胞培养中诱导焦点形成的效率与野生型没有显著差异。然而，与弱DNA结合剂相比，强DNA结合剂诱导的秦突变体的焦点形成速率和焦点大小更大。由cQin或v-Qin的翼状螺旋结构域组成的反式显性负构建体干扰了全长秦蛋白诱导的焦点形成。这些结果表明，DNA结合是秦蛋白转化的先决条件，强DNA结合与CEF中加速转化有关。
