Crawford A W, Michelsen J W, Beckerle M C
Department of Biology, University of Utah, Salt Lake City 84112.
J Cell Biol. 1992 Mar;116(6):1381-93. doi: 10.1083/jcb.116.6.1381.
Zyxin is an 82-kD protein first identified as a component of adhesion plaques and the termini of stress fibers near where they associate with the cytoplasmic face of the adhesive membrane. We report here that zyxin interacts with the actin cross-linking protein alpha-actinin. Zyxin cosediments with filamentous actin in an alpha-actinin-dependent manner and an association between zyxin and alpha-actinin is observed in solution by analytical gel filtration. The specificity of the interaction between zyxin and alpha-actinin was demonstrated by blot overlay experiments in which 125I-zyxin recognizes most prominently alpha-actinin among a complex mixture of proteins extracted from avian smooth muscle. By these blot overlay binding studies, we determined that zyxin interacts with the NH2-terminal 27-kD domain of alpha-actinin, a region that also contains the actin binding site. Solid phase binding assays were performed to evaluate further the specificity of the binding and to determine the affinity of the zyxin-alpha-actinin interaction. By these approaches we have demonstrated a specific, saturable, moderate-affinity interaction between zyxin and alpha-actinin. Furthermore, double-label immunofluorescence reveals that zyxin and alpha-actinin exhibit extensive overlap in their subcellular distributions in both chicken embryo fibroblasts and pigmented retinal epithelial cells. The significant colocalization of the two proteins is consistent with the possibility that the interaction between zyxin and alpha-actinin has a biologically relevant role in coordinating membrane-cytoskeletal interactions.
斑联蛋白是一种82-kD的蛋白质，最初被鉴定为粘着斑以及应力纤维末端（应力纤维在靠近与粘着膜细胞质面相连处）的组成成分。我们在此报告斑联蛋白与肌动蛋白交联蛋白α-辅肌动蛋白相互作用。斑联蛋白以α-辅肌动蛋白依赖的方式与丝状肌动蛋白共沉降，并且通过分析性凝胶过滤在溶液中观察到斑联蛋白与α-辅肌动蛋白之间存在关联。斑联蛋白与α-辅肌动蛋白之间相互作用的特异性通过印迹覆盖实验得以证明，在该实验中，125I-斑联蛋白在从禽平滑肌中提取的复杂蛋白质混合物中最显著地识别α-辅肌动蛋白。通过这些印迹覆盖结合研究，我们确定斑联蛋白与α-辅肌动蛋白的NH2末端27-kD结构域相互作用，该区域也包含肌动蛋白结合位点。进行了固相结合测定以进一步评估结合的特异性并确定斑联蛋白与α-辅肌动蛋白相互作用的亲和力。通过这些方法，我们证明了斑联蛋白与α-辅肌动蛋白之间存在特异性、可饱和的中等亲和力相互作用。此外，双标记免疫荧光显示，在鸡胚成纤维细胞和色素性视网膜上皮细胞中，斑联蛋白和α-辅肌动蛋白在亚细胞分布上有广泛重叠。这两种蛋白质的显著共定位与斑联蛋白和α-辅肌动蛋白之间的相互作用在协调膜-细胞骨架相互作用中具有生物学相关作用的可能性是一致的。
