Adhesion-dependent control of cyclin E/cdk2 activity and cell cycle progression in normal cells but not in Ha-ras transformed NRK cells.
Adhesion-dependent control of cyclin E/cdk2 activity and cell cycle progression in normal cells but not in Ha-ras transformed NRK cells.
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正常细胞中细胞周期蛋白 E/cdk2 活性和细胞周期进展的粘附依赖性控制，但 Ha-ras 转化的 NRK 细胞中则不然。
DOI：
10.1006/excr.1996.0346
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Fahl,WE
中科院分区：
文献类型：
--
作者：
Carstens,CP;Kramer,A;Fahl,WE
NRK 成纤维细胞失去对适当表面的粘附导致细胞周期停滞在 G1 晚期并且无法产生细胞周期蛋白 A。之前，我们表明细胞周期蛋白 A 的粘附依赖性表达受到转录调节。为了鉴定细胞周期蛋白 A 激活上游粘附介导的信号转导级联的元件，我们研究了粘附和悬浮 NRK 细胞中细胞周期蛋白 E 的表达及其相关激酶活性。发现细胞周期蛋白E的表达不受悬浮的影响。然而，从 G0 逮捕释放后 12 小时的 NRK 细胞制备的提取物中免疫沉淀的细胞周期蛋白 E 复合物被发现在悬浮细胞中没有催化活性，但在贴壁细胞中则不然。在用含有 G12V 突变的 c-Ha-rasoncogene 转化的 NRK 细胞中，没有观察到这种悬浮诱导的细胞周期蛋白 E 相关激酶活性抑制。当用细胞周期蛋白 A 启动子：荧光素酶报告基因构建体以及野生型 cdk2 或显性失活 cdk2 突变体的表达载体转染 G0 同步的 NRK 细胞时，发现细胞周期蛋白 A 的转录激活依赖于催化活性的 cdk2。细胞周期蛋白 E/cdk2 复合物的抑制通常归因于 cdk 抑制剂 p21Cip1 和 p27Kip1 的联合。然而，在p21Cip1或p27Kip1的表达或cdk2关联方面，贴壁细胞和悬浮细胞之间没有观察到差异，代谢标记细胞提取物的免疫沉淀物中也没有任何与cdk2或细胞周期蛋白E特异性相关的蛋白质。这些结果定义了粘附产生的信号通过调节细胞周期蛋白 E/cdk2 活性来控制细胞周期蛋白 A 表达的途径。
Loss of adhesion of NRK fibroblasts to an appropriate surface leads to cell cycle arrest in late G1and failure to produce cyclin A. Previously, we showed that adhesion-dependent expression of cyclin A is transcriptionally regulated. In an effort to identify elements of the adhesion-mediated signal transduction cascade upstream of cyclin A activation, we investigated the expression of cyclin E and its associated kinase activity in adherent and suspended NRK cells. Expression of cyclin E was found to be unaffected by suspension. However, cyclin E complexes immunoprecipitated from extracts prepared from NRK cells 12 h after release from G0arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibition of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-rasoncogene containing a G12V mutation. When G0-synchronized NRK cells were transfected with a cyclin A promoter:luciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional activation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attributed to association of the cdk inhibitors p21Cip1and p27Kip1. However, no differences between adherent and suspended cells could be observed for either expression or cdk2 association of p21Cip1or p27Kip1, nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. These results define a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity.