Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.
Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.
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DOI：
10.1016/s0021-9258(17)37011-4
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发表时间：
1994-03
期刊：
影响因子：
--
通讯作者：
E. Postel;C. Ferrone
中科院分区：
文献类型：
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作者：
E. Postel;C. Ferrone
nm 23基因编码的蛋白质参与肿瘤转移调节和各种基本细胞过程，尽管其机制尚不清楚。所有的Nm 23蛋白含有核苷二磷酸激酶（NDPK）的活性，其重要性，这些调节作用还不明显。人nm 23-H2基因的蛋白质产物在体外既作为核苷二磷酸激酶（NDPK-B; Gilles，A. M.，Presecan，E.，Vonica，A. Lascu，I.（1991）J.Biol.Chem.266，8784-8789）和作为转录因子（PuF; Postel，E. H、Berberich，S. J.，Flint，S. J.和Ferrone，C. A.（1993）Science 261，478-480）。为了了解这两种生化活性对NM 23-H2功能的意义，我们研究了NM 23-H2的DNA结合和转录活性与其NDPK功能之间的关系。使用编码NM 23-H2的cDNA的定点突变，我们创建了一个突变体，取代了氨基酸组氨酸118，该位点是磷酸酶中间体（不可磷酸化的氨基酸苯丙氨酸）形成中的假定磷酸化位点。H118 F突变蛋白被证明是无催化活性的，如在检测磷酸化酶中间体形成的放射性同位素测定和指示核苷二磷酸形成的偶联酶测定中所测量的。这些结果证实组氨酸118是NDPK-B活性的关键残基。此外，缺乏酶活性的H118 F突变蛋白在电泳迁移率变动分析中显示出对c-myc启动子的正常DNA结合亲和力，并且在体外使用c-myc基因保留了完整的转录活性。这些结果表明nm 23-H2的核苷二磷酸激酶活性一方面与其DNA结合和转录活性另一方面之间缺乏相关性，表明nm 23-H2基因编码双功能蛋白分子。
nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although the mechanisms remain undefined. All Nm23 proteins contain nucleoside diphosphate kinase (NDPK) activity whose significance to these regulatory effects is not yet evident. The protein product of the human nm23-H2 gene functions in vitro both as a nucleoside diphosphate kinase enzyme (NDPK-B; Gilles, A.-M., Presecan, E., Vonica, A. and Lascu, I. (1991) J. Biol. Chem. 266, 8784-8789) and as a transcription factor (PuF; Postel, E. H., Berberich, S. J., Flint, S. J. and Ferrone, C. A. (1993) Science 261, 478-480). To understand the significance of these two biochemical activities to NM23-H2 function, we have investigated the relationship between the DNA binding and transcriptional activity of NM23-H2 and its NDPK function. Using site-directed mutagenesis of the cDNA encoding NM23-H2, we have created a mutant substituting for the amino acid histidine 118, the presumed site of phosphorylation in the formation of the phosphoenzyme intermediate, the nonphosphorylatable amino acid phenylalanine. The H118F mutant protein is shown to be catalytically inactive as measured both in a radioisotopic assay that detects formation of the phosphorylated enzyme intermediate and in a coupled enzyme assay that indicates nucleoside diphosphate formation. These results confirm that histidine 118 is the critical residue for NDPK-B activity. In addition, the H118F mutant protein lacking enzymatic activity displayed normal DNA binding affinity for the c-myc promoter in electrophoretic mobility shift assays, and retained full transcriptional activity using the c-myc gene in vitro. These results indicate a lack of correlation between nucleoside diphosphate kinase activity of nm23-H2 on the one hand, and its DNA binding and transcriptional activity on the other, suggesting that the nm23-H2 gene encodes a bifunctional protein molecule.