The mechanism for regulation of the F-actin binding activity of IQGAP1 by calcium/calmodulin
The mechanism for regulation of the F-actin binding activity of IQGAP1 by calcium/calmodulin
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DOI：
10.1074/jbc.m109535200
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发表时间：
2002-04-05
影响因子：
4.8
通讯作者：
Bloom, GS
中科院分区：
文献类型：
--
作者：
Mateer, SC;McDaniel, AE;Bloom, GS
IQGAP 1与细胞皮质中的肌动蛋白丝共定位，并在体外与F-肌动蛋白和几种信号蛋白结合，包括钙调蛋白、Cdc 42、Rac 1和β-连环蛋白。人们认为IQGAP 1的F-肌动蛋白结合活性是通过其与这些信号分子的可逆结合来调节的，但其机制仍然不清楚。在这里，我们描述了钙调素的调节机制。发现纯化的肾上腺IQGAP 1由两个不同的蛋白池组成，其中一个结合F-肌动蛋白，缺乏钙调蛋白，另一个不结合F-肌动蛋白，但与钙调蛋白紧密相关。基于这一发现，我们假设钙调素负调节IQGAP 1与F-肌动蛋白的结合。使用重组野生型和突变IQGAP 1在体外以及在瞬时表达IQGAP 1-YFP的活细胞中检验了这一假设。在体外，野生型IQGAP 1对F-肌动蛋白的亲和力随着钙调素浓度的增加而降低，并且这种作用通过Ca 2+显著增强，并且需要IQGAP 1的IQ结构域。此外，我们发现，钙调素结合野生型IQGAPI更有效地在Ca 2+的存在下比EGTA，和所有8个IQ基序在每个IQGAP 1二聚体可以结合钙调素同时。在活细胞中，IQGAP 1-YFP定位于细胞皮质，但细胞内Ca 2+的升高可逆地诱导荧光融合蛋白成为弥漫性分布。总之，这些结果支持了一个模型，其中细胞内游离Ca 2+的升高促进钙调蛋白与IQGAP 1的结合，这反过来又抑制IQGAP 1与皮质肌动蛋白丝的结合。
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 Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAPI much more efficiently in the presence of Ca2+ 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 Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in f ree intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.