Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, University of Muenster, Von-Esmarch-Strasse 56, D-48149 Muenster, Germany.
J Biol Chem. 2011 Mar 4;286(9):7227-38. doi: 10.1074/jbc.M110.135095. Epub 2010 Dec 22.
Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 μM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.
S100 家族的 Ca(2+)-结合蛋白通过与 Ca(2+)负载构象中的特定细胞靶标结合并调节其来参与细胞内 Ca(2+)信号转导。由于不同 S100 蛋白的特定细胞靶标的信息仍然有限，我们开发了一种亲和方法，该方法仅选择与 S100 蛋白的生理活性二聚体结合的蛋白靶标。使用这种方法，我们在这里确定 IQGAP1 是 S100P 的一种新型和二聚体特异性靶标，S100P 是富含皮质细胞骨架的 S100 家族的成员。S100P 与 IQGAP1 的相互作用严格依赖于 Ca(2+)，解离常数为 0.2 μM。结合主要通过 IQGAP1 的 IQ 结构域和 S100P 的第一个 EF 手环发生，因此代表了 S100-靶标蛋白相互作用的新结构原理。在细胞刺激后，S100P 和 IQGAP1 共定位于或接近质膜，并且可以将复合物形成与 IQGAP1 的改变信号转导特性相关联。具体而言，EGF 诱导的 IQGAP1 的酪氨酸磷酸化被认为在质膜下区域的 IQGAP1 支架中组装信号中间物，在过表达 S100P 的细胞中显著减少，但在表达缺乏 IQGAP1 结合的 S100P 突变体的细胞中没有减少。此外，B-Raf 与 IQGAP1 的结合以及 EGF 触发的信号级联中 IQGAP1 下游的 MEK1/2 激活在升高的 S100P 水平下受损。因此，S100P 是 IQGAP1 的新型 Ca(2+)-依赖性调节剂，通过直接相互作用可以下调 IQGAP1 作为信号中间物的功能。
