Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration.
Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration.
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DOI：
10.1083/jcb.150.1.177
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发表时间：
2000-07-10
期刊：
影响因子：
--
通讯作者：
Chen YW
中科院分区：
文献类型：
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作者：
Bourguignon LY;Zhu H;Shao L;Chen YW
Tiam 1（T淋巴瘤侵袭和转移1）是Rho GTP酶（例如，Rac 1）并在乳腺肿瘤细胞中表达（例如，SP-1细胞系）。免疫沉淀和免疫印迹分析表明，Tiam 1和细胞骨架蛋白，锚蛋白，在体内作为一个复杂的物理关联。具体地，锚蛋白的锚蛋白重复结构域（ARD）负责Tiam 1结合。生化研究和缺失突变分析表明Tiam 1的氨基酸717和727之间的11个氨基酸序列（717 GEGTDAVKRS 727 L）是锚定结合结构域。最重要的是，与Tiam 1结合的锚蛋白激活Rho GTP酶上的GDP/GTP交换（例如，Rac 1）。使用大肠杆菌衍生的钙调素结合肽（CBP）标记的重组Tiam 1（氨基酸393-728）片段，包含锚蛋白结合结构域，我们已经检测到的Tiam 1（氨基酸393-738）片段和锚蛋白在体外之间的特异性结合相互作用。该Tiam 1片段还充当Tiam 1与锚蛋白结合的有效竞争性抑制剂。用Tiam 1 cDNA转染SP-1细胞刺激以下所有方面：（1）Tiam 1-锚蛋白在膜突起中的结合;（2）Rac 1活化;和（3）乳腺肿瘤细胞侵袭和迁移。用绿色荧光蛋白（GFP）标记的Tiam 1片段cDNA和Tiam 1 cDNA共转染SP1细胞，有效地阻断了Tiam 1-锚蛋白在细胞膜中的共定位，并通过锚蛋白相关的Tiam 1和肿瘤特异性表型抑制Rac 1上的GDP/GTP交换。这些研究结果表明，锚定Tiam 1相互作用在调节Rac 1信号和细胞骨架功能所需的致癌信号和转移性乳腺肿瘤细胞的进展中起着关键作用。
Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11–amino acid sequence between amino acids 717 and 727 of Tiam1 (717GEGTDAVKRS727L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1). Using an Escherichia coli–derived calmodulin-binding peptide (CBP)–tagged recombinant Tiam1 (amino acids 393–728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393–738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1–ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)–tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1–ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin–Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.