Buss F, Kendrick-Jones J, Lionne C, Knight A E, Côté G P, Paul Luzio J
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom.
J Cell Biol. 1998 Dec 14;143(6):1535-45. doi: 10.1083/jcb.143.6.1535.
Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.
肌球蛋白VI是一种非常规肌球蛋白，可能在真核细胞中通过富含肌动蛋白的细胞质区域参与囊泡膜运输。在本研究中，我们克隆并测序了编码鸡肠刷状缘肌球蛋白VI的cDNA。针对这种肌球蛋白VI的细菌表达片段制备了多克隆抗血清。亲和纯化的抗体通过免疫印迹和免疫沉淀对肌球蛋白VI具有高度特异性，并用于通过免疫荧光和免疫电子显微镜研究该蛋白的定位。结果发现，在NRK和A431细胞中，肌球蛋白VI与高尔基体以及细胞的前沿、皱褶边缘相关联，并且也存在于胞质池中。在通过表皮生长因子（EGF）刺激细胞表面产生皱褶的A431细胞中，肌球蛋白VI被磷酸化，并与埃兹蛋白和肌球蛋白V一起被募集到新形成的皱褶中。体外实验表明，一种p21激活激酶（PAK）可能是负责在运动结构域进行磷酸化的激酶。这些结果有力地支持了肌球蛋白VI在分泌和内吞途径的膜运输中发挥作用。
