Jeong Ha-Won, Li Zhigang, Brown Matthew D, Sacks David B
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
J Biol Chem. 2007 Jul 13;282(28):20752-62. doi: 10.1074/jbc.M700487200. Epub 2007 May 21.
IQGAP1 is a scaffolding protein involved in multiple fundamental cellular activities, including transcription, cell-cell attachment, and regulation of the cytoskeleton. To function in these pathways, IQGAP1 associates with numerous proteins such as actin, calmodulin, E-cadherin, beta-catenin, CLIP-170, and components of the mitogen-activated protein kinase pathway. Moreover, IQGAP1 binds to active Cdc42 and Rac1 but not RhoA or Ras. Here we show that IQGAP1 also binds to the small GTPase Rap1. In vitro analysis demonstrates a direct interaction between Rap1 and IQGAP1, which is augmented by activation (GTP loading) of Rap1. Cdc42 does not modulate the interaction between Rap1 and IQGAP1. In contrast, the association is eliminated by calmodulin both in the absence and presence of Ca(2+). The binding of Rap1 to a point mutant IQGAP1 construct that is unable to interact with calmodulin is 2.5-fold more than to wild type IQGAP1. Consistent with these findings, Rap1 binds to the IQ region of IQGAP1. Confocal microscopy demonstrates that Rap1 and IQGAP1 co-localize at the periphery of human epithelial cells but not in the cytoplasm. The interaction has functional sequelae. Overexpression of IQGAP1 substantially reduces adhesion-mediated activation of Rap1. In addition, Rap1 activation by cAMP is attenuated in cells that overexpress IQGAP1 and enhanced in cells lacking IQGAP1. These findings reveal that the interaction of IQGAP1 with Rap1 differs in several respects from its interaction with other small GTPases. Furthermore, our data suggest that IQGAP1 may link the calmodulin and Rap1 signaling pathways.
IQGAP1是一种支架蛋白，参与多种基本细胞活动，包括转录、细胞间附着以及细胞骨架调节。为在这些途径中发挥作用，IQGAP1与众多蛋白质相互作用，如肌动蛋白、钙调蛋白、E-钙黏蛋白、β-连环蛋白、CLIP-170以及丝裂原活化蛋白激酶途径的组分。此外，IQGAP1与活性Cdc42和Rac1结合，但不与RhoA或Ras结合。在此我们表明IQGAP1也与小GTP酶Rap1结合。体外分析证明Rap1与IQGAP1之间存在直接相互作用，这种相互作用因Rap1的激活（GTP加载）而增强。Cdc42不调节Rap1与IQGAP1之间的相互作用。相反，无论有无Ca(2+)，钙调蛋白均可消除这种相互作用。Rap1与无法与钙调蛋白相互作用的IQGAP1点突变构建体的结合比与野生型IQGAP1的结合多2.5倍。与这些发现一致，Rap1与IQGAP1的IQ区域结合。共聚焦显微镜检查表明Rap1和IQGAP1共定位于人上皮细胞的周边而非细胞质中。这种相互作用具有功能后果。IQGAP1的过表达显著降低黏附介导的Rap1激活。此外，cAMP介导的Rap1激活在过表达IQGAP1 的细胞中减弱，而在缺乏IQGAP1的细胞中增强。这些发现表明IQGAP1与Rap1的相互作用在几个方面不同于其与其他小GTP酶的相互作用。此外，我们的数据表明IQGAP1可能连接钙调蛋白和Rap1信号通路。
