Nuclear-targeted deleted in liver cancer 1 (DLC1) is less efficient in exerting its tumor suppressive activity both in vitro and in vivo.
Nuclear-targeted deleted in liver cancer 1 (DLC1) is less efficient in exerting its tumor suppressive activity both in vitro and in vivo.
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DOI：
10.1371/journal.pone.0025547
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Yam JW
中科院分区：
文献类型：
--
作者：
Chan LK;Ko FC;Sze KM;Ng IO;Yam JW
肝癌中的RhoA蛋白1（DLC 1）是一种重要的RhoGT 3激活蛋白（RhoGAP），它终止了人类癌症中的活性RhoA信号传导。越来越多的证据表明，DLC 1的肿瘤抑制活性不仅依赖于RhoGAP活性，而且还依赖于通过其与张力蛋白家族蛋白的相互作用而进行的适当的粘着斑定位。最近，有报道表明，DLC 1也可以在细胞核中发现;然而，核DLC 1的存在和相对的肿瘤抑制活性从未被清楚地解决。我们在此提供了新的证据表明，DLC 1蛋白，主要与局灶性粘连和定位在胞质溶胶，动态穿梭于细胞质和细胞核之间。用核输出阻断剂Leptomycin B（LMB）处理细胞，可将DLC 1保留在细胞核中。为了理解DLC 1的核进入，我们将DLC 1的氨基酸600-700鉴定为对其核定位重要的新区域。核DLC 1的肿瘤抑制活性通过使用具有优先核定位的DLC 1的核定位信号（NLS）融合变体（NLS-DLC 1）直接评估。在SMMC-7721 HCC细胞中，NLS-DLC 1的表达未能抑制体外集落形成和肌动蛋白应力纤维形成。通过皮下注射具有稳定NLS-DLC 1表达的p53−/− RasV 12成肝细胞，首次在体内证实了核DLC 1的肿瘤抑制活性被消除。当与非核靶向的DLC 1相比时，注射的具有NLS-DLC 1表达的成肝细胞有效地形成肿瘤。我们的研究确定了一个新的区域负责DLC 1的核进入，并证明了DLC 1在不同的细胞室在体外和体内的功能差异。
Deleted in liver cancer 1 (DLC1) serves as an important RhoGTPase activating protein (RhoGAP) protein that terminates active RhoA signaling in human cancers. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends not only on RhoGAP activity, but also relies on proper focal adhesion localization through its interaction with tensin family proteins. Recently, there are reports showing that DLC1 can also be found in the nucleus; however, the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed. We herein provide new evidence that DLC1 protein, which predominantly associated with focal adhesions and localized in cytosol, dynamically shuttled between cytoplasm and nucleus. Treatment of cells with nuclear export blocker, Leptomycin B (LMB), retained DLC1 in the nucleus. To understand the nuclear entry of DLC1, we identified amino acids 600–700 of DLC1 as a novel region that is important for its nuclear localization. The tumor suppressive activity of nuclear DLC1 was directly assessed by employing a nuclear localization signal (NLS) fusion variant of DLC1 (NLS-DLC1) with preferential nuclear localization. In SMMC-7721 HCC cells, expression of NLS-DLC1 failed to suppress colony formation and actin stress fiber formation in vitro. The abrogated tumor suppressive activity of nuclear DLC1 was demonstrated for the first time in vivo by subcutaneously injecting p53−/− RasV12 hepatoblasts with stable NLS-DLC1 expression in nude mice. The injected hepatoblasts with NLS-DLC1 expression effectively formed tumors when compared with the non-nuclear targeted DLC1. Our study identified a novel region responsible for the nuclear entry of DLC1 and demonstrated the functional difference of DLC1 in different cellular compartments both in vitro and in vivo.
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