Overexpression of kinase suppressor of Ras upregulates the high-molecular-weight tropomyosin isoforms in ras-transformed NIH 3T3 fibroblasts
Overexpression of kinase suppressor of Ras upregulates the high-molecular-weight tropomyosin isoforms in ras-transformed NIH 3T3 fibroblasts
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DOI：
10.1128/mcb.23.5.1786-1797.2003
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发表时间：
2003-03-01
影响因子：
5.3
通讯作者：
Morrison, DK
中科院分区：
文献类型：
--
作者：
Janssen, RAJ;Kim, PN;Morrison, DK
原肌球蛋白(TM)高相对分子质量异构体的下调被认为是细胞转化过程中的一个重要事件。在RAS转化的成纤维细胞中，TM的抑制依赖于Raf-1激酶的活性；然而，对RAS的其他下游效应因子，如MEK和ERK的需求则不太清楚。在本研究中，我们利用Ras的丝裂原活化蛋白激酶支架蛋白激酶抑制因子(KSR)来进一步研究TM的调控，并阐明MEK/ERK信号在这一过程中的重要性。在这里，我们报告了野生型KSR1在ras转化的成纤维细胞中的过表达可以恢复TM的表达，并诱导细胞扁平和应力纤维的形成。此外，我们发现在ras转化的细胞中，TM-α启动子的转录活性降低，并且KSR1恢复TM的过程与该启动子的转录增加相一致。尽管ERK活性在过度表达KSR1的细胞中被抑制，但仅抑制ERK不足以上调TM的表达。KSR1介导的应力纤维形成和TM转录效应需要岩石激酶的活性，因为这些效应可以被岩石抑制剂Y27632抑制。KSR1的过表达并不直接调节岩石的活性，但确实允许岩石与肌动蛋白聚合机制的重新连接。最后，KSR1诱导的所有效应都是由KSR1的C末端结构域介导的，并且依赖于KSR-MEK的相互作用。
The down-regulation of the high-molecular-weight isoforms of tropomyosin (TM) is considered to be an essential event in cellular transformation. In ras-transformed fibroblasts, the suppression of TM is dependent on the activity of the Raf-1 kinase; however, the requirement for other downstream effectors of Ras, such as MEK and ERK, is less clear. In this study, we have utilized the mitogen-activated protein kinase scaffolding protein Kinase Suppressor of Ras (KSR) to further investigate the regulation of TM and to clarify the importance of MEK/ERK signaling in this process. Here, we report that overexpression of wild-type KSR1 in ras-transformed fibroblasts restores TM expression and induces cell flattening and stress fiber formation. Moreover, we find that the transcriptional activity of a TM-alpha promoter is decreased in ras-transformed cells and that the restoration of TM by KSR1 coincides with increased transcription from this promoter. Although ERK activity was suppressed in cells overexpressing KSR1, ERK inhibition alone was insufficient to upregulate TM expression. The KSR1-mediated effects on stress fiber formation and TM transcription required the activity of the ROCK kinase, because these effects could be suppressed by the ROCK inhibitor, Y27632. Overexpression of KSR1 did not directly regulate ROCK activity, but did permit the recoupling of ROCK to the actin polymerization machinery. Finally, all of the KSR1-induced effects were mediated by the C-terminal domain of KSR1 and were dependent on the KSR-MEK interaction.