Department of Biochemistry, School of Biology, Moscow State University, Moscow 119991, Russian Federation.
Arch Biochem Biophys. 2012 May;521(1-2):62-70. doi: 10.1016/j.abb.2012.03.010. Epub 2012 Mar 19.
It has been previously reported that phosphorylated cofilin interacted with 14-3-3ζ protein to generate a sub-micromolar K(d) binary complex. Here we challenge this hypothesis by analyzing the direct association of recombinant cofilin with 14-3-3ζ using different in vitro biochemical methods. Phosphorylated cofilin at high concentration binds to 14-3-3 immobilized on nitrocellulose, however no complex formation was detected by means of native gel electrophoresis or chemical crosslinking. Intact dimeric or mutant monomeric 14-3-3 was unable to form stable complexes with phosphorylated or unphosphorylated cofilin detected by size-exclusion chromatography. In co-sedimentation assay 14-3-3 did not affect interaction of cofilin with F-actin. The data of native gel electrophoresis indicate that 14-3-3 did not affect interaction of cofilin with G-actin. Thus, cofilin only weakly interacts with 14-3-3 and therefore cannot directly compete with phosphorylated small heat shock protein HspB6 for its binding to 14-3-3. It is hypothesized that phosphorylated HspB6 might affect interaction of 14-3-3 with protein phosphatases (and/or protein kinases) involved in dephosphorylation (or phosphorylation) of cofilin and by this means regulate cofilin-dependent reorganization of cytoskeleton.
先前有报道称，磷酸化丝切蛋白与 14-3-3ζ 蛋白相互作用，生成亚毫摩尔 K(d) 二元复合物。在这里，我们通过使用不同的体外生化方法分析重组丝切蛋白与 14-3-3ζ 的直接关联，对这一假说提出了挑战。高浓度磷酸化丝切蛋白与固定在硝酸纤维素上的 14-3-3 结合，但通过天然凝胶电泳或化学交联均未检测到复合物的形成。完整的二聚体或突变的单体 14-3-3 不能与通过大小排阻层析检测到的磷酸化或非磷酸化丝切蛋白形成稳定的复合物。在共沉淀测定中，14-3-3 不影响丝切蛋白与 F-肌动蛋白的相互作用。天然凝胶电泳的数据表明，14-3-3 不影响丝切蛋白与 G-肌动蛋白的相互作用。因此，丝切蛋白与 14-3-3 的相互作用较弱，因此不能直接与磷酸化的小分子热休克蛋白 HspB6 竞争与 14-3-3 的结合。据推测，磷酸化的 HspB6 可能会影响 14-3-3 与参与丝切蛋白去磷酸化（或磷酸化）的蛋白磷酸酶（和/或蛋白激酶）的相互作用，并通过这种方式调节丝切蛋白依赖的细胞骨架重组。
