Mateer Scott C, McDaniel Amanda E, Nicolas Valerie, Habermacher Geoffrey M, Lin Mei-Jung Sun, Cromer Damond A, King Michelle E, Bloom George S
Departments of Biology and Cell Biology, University of Virginia, Charlottesville, Virginia 22903, USA.
J Biol Chem. 2002 Apr 5;277(14):12324-33. doi: 10.1074/jbc.M109535200. Epub 2002 Jan 24.
IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and beta-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca(2+) and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca(2+) than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca(2+) reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca(2+) promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.
IQGAP1与肌动蛋白丝在细胞皮层中共定位，并在体外与F-肌动蛋白以及几种信号蛋白结合，包括钙调蛋白、Cdc42、Rac1和β-连环蛋白。据认为，IQGAP1的F-肌动蛋白结合活性受其与这些信号分子的可逆结合调节，但其机制仍不清楚。在这里，我们描述了钙调蛋白的调节机制。发现纯化的肾上腺IQGAP1由两个不同的蛋白池组成，其中一个与F-肌动蛋白结合且缺乏钙调蛋白，另一个不与F-肌动蛋白结合但与钙调蛋白紧密结合。基于这一发现，我们假设钙调蛋白负向调节IQGAP1与F-肌动蛋白的结合。使用重组野生型和突变型IQGAP1在体外以及在瞬时表达IQGAP1-YFP的活细胞中对这一假设进行了测试。在体外，野生型IQGAP1对F-肌动蛋白的亲和力随着钙调蛋白浓度的增加而降低，并且这种效应在Ca(2+)存在下显著增强，并且需要IQGAP1的IQ结构域。此外，我们发现钙调蛋白在Ca(2+)存在下比在EGTA存在下更有效地结合野生型IQGAP1，并且每个IQGAP1二聚体中的所有8个IQ基序可以同时结合钙调蛋白。在活细胞中，IQGAP1-YFP定位于细胞皮层，但细胞内Ca(2+)的升高可逆地诱导荧光融合蛋白变得弥散分布。综上所述，这些结果支持了一个模型，即细胞内游离Ca(2+)的升高促进钙调蛋白与IQGAP1的结合，这反过来又抑制IQGAP1与皮层肌动蛋白丝的结合。
