Site-directed mutagenesis of nm23-H1 - Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells
Site-directed mutagenesis of nm23-H1 - Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells
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DOI：
10.1074/jbc.271.41.25107
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发表时间：
1996-10-11
影响因子：
4.8
通讯作者：
Steeg, PS
中科院分区：
文献类型：
--
作者：
MacDonald, NJ;Freije, JMP;Steeg, PS
我们使用定点诱变和体外肿瘤细胞运动测定首次报道了 Nm23 序列与其肿瘤转移抑制能力的相关性。 MDA-MB-435人乳腺癌细胞用对照表达载体(pCMVB-amneo)转染，该载体含有野生型nm23-H1，或编码以下氨基酸突变的nm23-H1载体：丝氨酸44，磷酸化位点；脯氨酸 96，果蝇 nm23 同源物中的 k-pn 突变，导致发育缺陷；组氨酸 118，参与 Nm23 的核苷二磷酸激酶活性；丝氨酸120，人类神经母细胞瘤中的突变和磷酸化位点。与对照载体转染子相比，野生型nm23-H1转染子对血清的运动性降低44-98％，对自分泌运动蛋白的运动性降低86-99％。脯氨酸 96 k-pn、丝氨酸 120 变为甘氨酸以及较小程度的丝氨酸 120 变为丙氨酸突变型 nm23-H1 转染细胞系表现出等于或高于对照转染子的运动水平，表明这些突变可以消除 nm23-H1 的运动抑制表型。没有观察到对细胞增殖的影响，丝氨酸 44 至丙氨酸 nm23-H1 突变转染子也没有运动，证明了数据的特异性。这些数据确定了 nm23-H1 影响其转移抑制作用的第一个结构基序，并表明 Nm23 抑制途径中存在复杂的生化关联或活性。
We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVB-amneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway.