Association of JNK1 with p21waf1 and p53: modulation of JNK1 activity.
Association of JNK1 with p21waf1 and p53: modulation of JNK1 activity.
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JNK1 与 p21waf1 和 p53 的关联：JNK1 活性的调节。
DOI：
10.1002/mc.10096
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Pelling,JillC
中科院分区：
文献类型：
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作者：
Xue,Yue;Ramaswamy,NadjaT;Hong,Xiaoman;Pelling,JillC
哺乳动物细胞暴露于遗传毒性应激导致c-jun氨基末端激酶（JNK）-应激激活蛋白激酶（SAPK）途径活化，并诱导DNA修复酶和细胞周期调节蛋白（如p53和p21 waf 1）。p53肿瘤抑制蛋白传递激活p21 waf 1基因表达的信号。p21 waf 1蛋白然后限制细胞周期进程，从而允许DNA修复发生的时间。在这项研究中，我们研究了野生型和突变型p53蛋白水平的调节对A1 - 5大鼠成纤维细胞系中基础JNK 1活性的影响。该细胞系含有编码温度敏感性p53蛋白的ap 53基因，这使我们能够调节细胞中产生的野生型和突变型p53蛋白的相对水平。使用免疫复合物激酶测定来测量JNK 1活性，我们证明表达野生型构象p53蛋白的细胞（当在32.5°C下生长时）表现出非常低水平的JNK 1活性。当细胞在37°C或39°C生长以主要表达突变型p53蛋白时，JNK 1活性的基础水平显著高于32.5°C。我们还证明了该细胞系中p53，p21 waf 1和JNK 1蛋白之间的蛋白质相互作用。野生型p53蛋白（在32.5°C下表达）和突变型p53 val 135蛋白（在37°C和39°C下表达）均存在于JNK 1蛋白的免疫复合物中。在存在野生型p53蛋白以诱导p21 waf 1表达的条件下（在32.5°C下），在JNK 1免疫复合物中也检测到比在37°C和39°C下更高水平的p21 waf 1蛋白。我们接下来研究了p53蛋白和p21 waf 1蛋白的共缔合对JNK 1活性的影响。我们测量了表达野生型p53和p21 waf 1的细胞或p21 waf 1缺失细胞中JNK 1活性的基础水平，并证明与p21 waf 1缺失细胞相比，表达p53和p21 waf 1蛋白的细胞显示出约3倍的JNK 1活性基础水平。为了证实细胞中p21 waf 1蛋白表达导致JNK 1活性降低，我们用ap 21 waf 1表达载体转染p21 waf 1 −/−细胞。我们观察到外源性p21 waf 1蛋白在这些细胞中表达后，JNK 1活性受到抑制。我们的研究结果为p53和p21 waf 1蛋白调节JNK 1通路提供了证据，并支持JNK 1活性调节通过JNK 1，p53和p21 waf 1蛋白之间的蛋白质相互作用发生的假设。© 2002 Wiley利斯公司
Exposure of mammalian cells to genotoxic stress results in activation of the c‐jun amino‐terminal kinase (JNK)‐stress‐activated protein kinase (SAPK) pathway and induction of DNA repair enzymes and cell cycle–regulatory proteins such as p53 andp21waf1. The p53 tumor suppressor protein transmits signals that activatep21waf1gene expression. The p21waf1 protein then restricts cell‐cycle progression, thereby allowing time for DNA repair to occur. In this study, we investigated the effects of modulation of the level of wild‐type and mutantp53protein on basal JNK1 activity in the A1‐5 rat fibroblast cell line. This cell line contains ap53gene coding for a temperature‐sensitive p53 protein, which allows us to regulate the relative level of wild‐type and mutant p53 protein produced in cells. Using the immune complex kinase assay to measure JNK1 activity, we demonstrated that cells expressing the wild‐type–conformation p53 protein (when grown at 32.5°C) exhibited a very low level of JNK1 activity. When cells were grown at 37°C or 39°C to express predominantly mutant p53 protein, basal level of JNK1 activity was significantly higher than at 32.5°C. We also demonstrated protein‐protein interactions between the p53, p21waf1, and JNK1 proteins in this cell line. Both wild‐type p53 protein (expressed at 32.5°C) and mutant p53val135protein (expressed at 37°C and 39°C) were present in immunocomplexes of JNK1 protein. Under conditions where wild‐type p53 protein was present to induce p21waf1 expression (at 32.5°C), a higher level of p21waf1 protein was also detected in the JNK1 immunocomplexes than in those at 37°C and 39°C. We next investigated the effect that co‐association of p53 protein and p21waf1 protein would have on JNK1 activity. We measured basal levels of JNK1 activity in cells expressing wild‐type p53 and p21waf1, or inp21waf1‐null cells, and demonstrated that cells expressing both p53 and p21waf1 proteins exhibited an approximately threefold lower basal level of JNK1 activity when compared withp21waf1‐null cells. To confirm that p21waf1 protein expression in cells resulted in reduced JNK1 activity, we transfectedp21waf1−/−cells with ap21waf1expression vector. We observed that JNK1 activity was inhibited after exogenous p21waf1 protein was expressed in these cells. Our results provide evidence for modulation of the JNK1 pathway by p53 and p21waf1 proteins and support the hypothesis that modulation of JNK1 activity occurred through protein‐protein interactions between JNK1, p53, and p21waf1 proteins. © 2002 Wiley‐Liss, Inc.