Chen Yu, Takizawa Norio, Crowley Jessica L, Oh Sang W, Gatto Cheryl L, Kambara Taketoshi, Sato Osamu, Li Xiang-Dong, Ikebe Mitsuo, Luna Elizabeth J
Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
J Biol Chem. 2003 Nov 14;278(46):46094-106. doi: 10.1074/jbc.M305311200. Epub 2003 Aug 12.
Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.
抗去污剂膜含有信号蛋白和整合膜蛋白，这些蛋白组织形成了称为脂筏的富含胆固醇的结构域。由于与包括肌动蛋白、肌球蛋白II、肌球蛋白1G、血影蛋白以及一种名为 supervillin 的肌动蛋白和膜结合蛋白相关联，这些抗去污剂膜的一个子集（DRM-H）表现出更高的浮力密度（约1.16 g/ml）（内布尔，T.，佩斯顿贾马斯普，K. N.，莱齐克，J. D.，克劳利，J. L.，吴，S. W.，和卢娜，E. J.（2002年）《生物化学杂志》277卷，43399 - 43409页）。为了表征DRM-H细胞骨架蛋白之间的相互作用，我们研究了新型supervillin N端（具体为氨基酸1 - 830）的结合伙伴。我们发现supervillin N端直接与肌球蛋白II以及F-肌动蛋白结合。三个F-肌动蛋白结合位点被定位到大约280 - 342、大约344 - 422和大约700 - 830氨基酸序列内。具有这些位点组合的序列促进F-肌动蛋白交联和/或成束。Supervillin氨基酸1 - 174与鸡胃肌球蛋白和非肌肉肌球蛋白IIA（MYH-9）中的S2结构域特异性相互作用，但与骨骼肌肌球蛋白II的结合很少。还观察到与细丝蛋白的直接或间接结合。在COS7细胞中过表达supervillin氨基酸1 - 174会破坏肌球蛋白IIB的定位，而对肌动蛋白丝没有明显影响。综上所述，这些结果表明supervillin可能在富含胆固醇的膜结构域介导肌动蛋白和肌球蛋白II丝的组织。
