ALTERNATE BINDING OF ACTIN AND CALMODULIN TO MULTIPLE SITES ON DYSTROPHIN
ALTERNATE BINDING OF ACTIN AND CALMODULIN TO MULTIPLE SITES ON DYSTROPHIN
复制标题
DOI：
10.1074/jbc.270.10.5578
复制
发表时间：
1995-03-10
影响因子：
4.8
通讯作者：
FOSTER, JL
中科院分区：
文献类型：
--
作者：
JARRETT, HW;FOSTER, JL
小鼠肌营养不良蛋白蛋白序列1-385和各种缺失突变体在大肠杆菌中表达为融合蛋白，并表征肌动蛋白、钙调蛋白和肌钙蛋白C的结合。含有融合蛋白的序列1-385以129 +/- 65 nM的表观解离常数结合肌动蛋白，如使用固相免疫测定法测量的。超离心共沉淀试验和生物素化肌动蛋白结合试验也观察到高亲和力。缺失突变体的结果和基于序列同源性的分析与肌营养不良蛋白的该区域内序列位置18-37（ABS 1）、128-149（ABS 2）处的两个或三个高亲和力F-肌动蛋白结合序列一致，并且可能在称为ABS 3的新区域（86-120）处，缺乏这些序列但含有肌养蛋白三螺旋序列的融合蛋白也结合肌动蛋白，但亲和力降低。依赖的方式和竞争性抑制F-肌动蛋白结合。结果与肌营养不良蛋白的该区域中大约序列位置18-42（CBS 1）和104-125（CBS 2）处的两个Ca 2+钙调蛋白结合位点一致，钙调蛋白亲和力分别为2.1 +/- 1和1.6 +/- 1.2 μ M。肌钙蛋白C可以替代钙调蛋白，尽管其结合亲和力低约2倍。这些结果表明，钙调蛋白（或肌钙蛋白C）结合交替，并可能调节F-肌动蛋白结合肌营养不良蛋白已经假定为其他细胞骨架蛋白是同源的肌营养不良蛋白。
Mouse dystrophin protein sequence 1-385 and various deletion mutants were expressed in Escherichia coli as fusion proteins, and the binding of actin, calmodulin, and troponin C were characterized. The fusion protein-containing sequence 1-385 bound actin with an apparent dissociation constant of 129 +/- 65 nM as measured using a solid-phase immunoassay. High affinity was also observed with ultracentrifuge cosedimentation assays and biotinylated-actin binding assays. Results with deletion mutants and analysis based upon sequence homology were consistent with two or three high affinity F-actin-binding sequences within this region of dystrophin at sequence positions 18-37 (ABS 1), 128-149 (ABS 2), and potentially at a new region called ABS 3 (86-120), A fusion protein lacking these sequences but containing dystrophin triple helix sequences also bound actin but with reduced affinity.Calmodulin binds to dystrophin sequence 1-385 in a Ca2+-dependent manner and competitively inhibits F-actin binding. Results were consistent with two Ca2+ calmodulin-binding sites in this region of dystrophin at approximate sequence positions 18-42 (CBS 1) and 104-125 (CBS 2) with calmodulin affinities of 2.1 +/- 1 and 1.6 +/- 1.2 mu M, respectively. Troponin C can substitute for calmodulin, although it binds with about 2-fold lower affinity. These results suggest that calmodulin (or troponin C) binding alternates with and may regulate F-actin binding by dystrophin much as has been postulated for other cytoskeletal proteins which are homologous to dystrophin.