Regulation of Focal Adhesion Kinase Activation, Breast Cancer Cell Motility, and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1
Regulation of Focal Adhesion Kinase Activation, Breast Cancer Cell Motility, and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1
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DOI：
10.1128/mcb.00175-13
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发表时间：
2013-07-01
影响因子：
5.3
通讯作者：
Frost, Jeffrey A.
中科院分区：
文献类型：
--
作者：
Carr, Heather S.;Zuo, Yan;Frost, Jeffrey A.
Net1是一种RhoA鸟嘌呤核苷酸交换因子，在人类癌症的一个亚群中过表达，并在体外促进癌细胞的运动和侵袭。然而，它在细胞运动和侵袭中的作用的分子机制还没有被描述。在目前的工作中，我们证明了两种Net1亚型在乳腺癌细胞中的表达是有效的细胞运动所必需的。虽然Net1亚型表达的缺失只部分阻断了RhoA的激活，但它抑制溶血磷脂酸(LPA)刺激的迁移的效果与敲除RhoA本身一样有效。然而，我们证明，Net1A亚型主要控制肌球蛋白轻链的磷酸化，并在迁移过程中需要后缘回缩。Net1A与粘着斑激酶(FAK)相互作用，定位于粘着斑处，在细胞扩散过程中是FAK激活和粘着点成熟所必需的。Net1A表达也是通过Matrigel矩阵有效侵袭所必需的。对侵袭细胞的分析表明，Net1a是阿米巴侵袭所必需的，Net1a的表达缺失会导致细胞向间充质表型转变，其特征是高β(1)整合素活性和膜型1基质金属蛋白酶(MT1-MMPs)的表达。这些结果表明，Net1A异构体在控制平面细胞运动中的FAK激活和细胞外基质(ECM)侵袭过程中的阿米巴运动中所起的作用是以前未知的。
Net1 is a RhoA guanine nucleotide exchange factor (GEF) that is overexpressed in a subset of human cancers and contributes to cancer cell motility and invasion in vitro. However, the molecular mechanism accounting for its role in cell motility and invasion has not been described. In the present work, we show that expression of both Net1 isoforms in breast cancer cells is required for efficient cell motility. Although loss of Net1 isoform expression only partially blocks RhoA activation, it inhibits lysophosphatidic acid (LPA)-stimulated migration as efficiently as knockdown of RhoA itself. However, we demonstrate that the Net1A isoform predominantly controls myosin light-chain phosphorylation and is required for trailing edge retraction during migration. Net1A interacts with focal adhesion kinase (FAK), localizes to focal adhesions, and is necessary for FAK activation and focal adhesion maturation during cell spreading. Net1A expression is also required for efficient invasion through a Matrigel matrix. Analysis of invading cells demonstrates that Net1A is required for amoeboid invasion, and loss of Net1A expression causes cells to shift to a mesenchymal phenotype characterized by high beta(1)-integrin activity and membrane type 1 matrix metalloproteinase (MT1-MMP) expression. These results demonstrate a previously unrecognized role for the Net1A isoform in controlling FAK activation during planar cell movement and amoeboid motility during extracellular matrix (ECM) invasion.