García-Palmero Irene, Pompas-Veganzones Noemí, Villalobo Eduardo, Gioria Sophie, Haiech Jacques, Villalobo Antonio
Department of Cancer Biology, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Spain.
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Spain.
FEBS Lett. 2017 Apr;591(8):1176-1186. doi: 10.1002/1873-3468.12623. Epub 2017 Mar 26.
We identified the Grb7 family members, Grb10 and Grb14, as Ca -dependent CaM-binding proteins using Ca -dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7.
我们使用钙依赖的钙调蛋白亲和层析法，像之前对Grb7所做的那样，鉴定出Grb7家族成员Grb10和Grb14是钙依赖的钙调蛋白结合蛋白。使用与这些位点对应的荧光标记肽来鉴定潜在的钙调蛋白结合位点并进行实验测试。还测定了这些肽对钙调蛋白的表观亲和常数，以及有效结合这些肽所需的与钙调蛋白结合的钙离子的最小数量。我们制备了缺乏已鉴定位点的三种衔接蛋白的缺失突变体，并确定它们按照Grb7＞＞Grb14＞Grb10的顺序丧失或显著降低了它们的钙调蛋白结合能力。与Grb7中存在的单个钙调蛋白结合位点相比，Grb10和Grb14中似乎存在不止一个钙调蛋白结合位点和/或辅助钙调蛋白结合位点。
