Briggs Michael W, Li Zhigang, 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. 2002 Mar 1;277(9):7453-65. doi: 10.1074/jbc.M104315200. Epub 2001 Dec 4.
Beta-catenin, an oncoprotein integral to cell-cell adhesion and proliferative signaling, is increased in several malignancies. The recently discovered calmodulin-binding protein IQGAP1 binds stoichiometrically to beta-catenin and regulates the association of beta-catenin with the cell-cell adhesion complex. In the present work, we investigated the role of IQGAP1 on the transcriptional co-activator function of beta-catenin, and whether calmodulin modulated the functional interaction between IQGAP1 and beta-catenin. In vitro competition assays revealed that both Ca(2+)/calmodulin and apo-calmodulin, when pre-bound to IQGAP1, prevented binding of beta-catenin to IQGAP1, and partially displaced beta-catenin pre-bound to IQGAP1 when added subsequently. Conversely, beta-catenin partially displaced apo-calmodulin, but not Ca(2+)/calmodulin, from IQGAP1. Overexpression of IQGAP1 in SW480 colon carcinoma cells enhanced beta-catenin-mediated transcriptional co-activation by 1.72-fold, and this stimulation was significantly attenuated upon antagonism of calmodulin using the cell-permeable antagonist CGS9343B. Moreover, an IQGAP1 mutant that does not bind calmodulin was unable to stimulate beta-catenin transcriptional function. Results of pulse-chase analyses suggested that IQGAP1 slowed the turnover of soluble, but not total, beta-catenin. Immunocytochemistry revealed that IQGAP1 overexpression increased the amount of beta-catenin located in the nucleus, whereas incubation of cells with CGS9343B blocked this accumulation. Together, our results imply that IQGAP1 enhances the function of beta-catenin in the nucleus and that calmodulin regulates this stimulation.
β-连环蛋白是一种对细胞间黏附和增殖信号至关重要的癌蛋白，在多种恶性肿瘤中表达增加。最近发现的钙调蛋白结合蛋白IQGAP1与β-连环蛋白以化学计量比结合，并调节β-连环蛋白与细胞间黏附复合物的结合。在本研究中，我们研究了IQGAP1对β-连环蛋白转录共激活功能的作用，以及钙调蛋白是否调节IQGAP1与β-连环蛋白之间的功能相互作用。体外竞争试验表明，预先与IQGAP1结合的Ca(2+)/钙调蛋白和脱辅基钙调蛋白均可阻止β-连环蛋白与IQGAP1的结合，随后添加时可部分取代预先与IQGAP1结合的β-连环蛋白。相反，β-连环蛋白可部分取代IQGAP1上的脱辅基钙调蛋白，但不能取代Ca(2+)/钙调蛋白。在SW480结肠癌细胞中过表达IQGAP1可使β-连环蛋白介导的转录共激活增强1.72倍，使用细胞可渗透拮抗剂CGS9343B拮抗钙调蛋白后，这种刺激作用明显减弱。此外，一种不与钙调蛋白结合的IQGAP1突变体无法刺激β-连环蛋白的转录功能。脉冲追踪分析结果表明，IQGAP1减缓了可溶性β-连环蛋白的周转，但不影响总β-连环蛋白的周转。免疫细胞化学显示，IQGAP1过表达增加了细胞核中β-连环蛋白的含量，而用CGS9343B处理细胞可阻断这种积累。总之，我们的结果表明IQGAP1增强了β-连环蛋白在细胞核中的功能，并且钙调蛋白调节这种刺激作用。
