Guillemot Laurent, Paschoud Serge, Jond Lionel, Foglia Andrea, Citi Sandra
Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland.
Mol Biol Cell. 2008 Oct;19(10):4442-53. doi: 10.1091/mbc.e08-06-0558. Epub 2008 Jul 23.
Small GTPases control key cellular events, including formation of cell-cell junctions and gene expression, and are regulated by activating and inhibiting factors. Here, we characterize the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2. During tight junction assembly by the calcium-switch, Rac1 shows two transient peaks of activity, at earlier (10-20 min) and later (3-8 h) time points. Paracingulin depletion reduces such peaks of Rac1 activation in a Tiam1-dependent manner, resulting in a delay in junction formation. Paracingulin physically interacts with GEF-H1 and Tiam1 in vivo and in vitro, and it is required for their efficient recruitment to junctions, based on immunofluorescence and biochemical experiments. Our results provide the first description of a junctional protein that interacts with GEFs for both Rac1 and RhoA, and identify a novel molecular mechanism whereby Rac1 is activated during junction formation.
小GTP酶控制关键的细胞活动，包括细胞间连接的形成和基因表达，并受激活因子和抑制因子的调控。在此，我们将连接蛋白副连环蛋白（paracingulin）描述为一种新型的小GTP酶Rac1和RhoA活性的调节因子，它通过与其各自的激活因子Tiam1和GEF-H1的功能相互作用来发挥作用。在汇合的上皮单层细胞中，副连环蛋白的缺失导致RhoA活性增加以及紧密连接蛋白claudin-2的mRNA表达增加。在通过钙转换进行紧密连接组装的过程中，Rac1在较早（10 - 20分钟）和较晚（3 - 8小时）的时间点出现两个活性的瞬时峰值。副连环蛋白的缺失以Tiam1依赖的方式降低了Rac1激活的这些峰值，导致连接形成延迟。基于免疫荧光和生化实验，副连环蛋白在体内和体外均与GEF-H1和Tiam1发生物理相互作用，并且其有效募集到连接处是必需的。我们的结果首次描述了一种与Rac1和RhoA的鸟嘌呤核苷酸交换因子（GEF）相互作用的连接蛋白，并确定了一种在连接形成过程中激活Rac1的新分子机制。
