Deguen B, Mérel P, Goutebroze L, Giovannini M, Reggio H, Arpin M, Thomas G
Laboratoire de Génétique des Tumeurs, INSERM U434, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Hum Mol Genet. 1998 Feb;7(2):217-26. doi: 10.1093/hmg/7.2.217.
Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.
尽管神经纤维瘤病2型基因的产物施万蛋白与定义ERM家族的三种细胞骨架与膜蛋白连接体具有同源性，但其发挥肿瘤抑制活性的机制仍不清楚。基于对自然发生突变的了解，开展了施万蛋白的功能研究。将编码两种野生型异构体和九种突变形式的构建体转染到HeLa细胞中。瞬时表达的野生型异构体均在质膜下方被观察到。在此位置，它们不溶于去污剂，并通过细胞松弛素D处理重新分布，表明与基于肌动蛋白的细胞骨架结构相互作用。在第219和220位有单氨基酸取代的蛋白质表现出相同的特性。三种不同的截短施万蛋白是大多数自然发生的NF2突变的原型，它们的位置和对细胞松弛素D的敏感性均未受影响。然而，它们被发现可溶于去污剂，表明与基于肌动蛋白的结构的相互作用较松散。在蛋白质C端一半的第360位有单氨基酸取代的突变体也观察到溶解性增加。在第118位有单氨基酸缺失或在N端结构域内有83个氨基酸缺失的突变蛋白失去了膜下定位，并倾向于聚集在不受细胞松弛素D处理影响的核周斑块中。当N端结构域完全缺失时也观察到类似的行为。综上所述，这些观察结果表明，N端结构域是将该蛋白定位在膜上的主要决定因素，在膜上它与基于肌动蛋白的细胞骨架结构弱相互作用。C端结构域增强了这种相互作用。除极少数例外，大多数失去肿瘤抑制活性的自然发生的突变施万蛋白在涉及基于肌动蛋白的结构的相互作用中受损，并且不再牢固地维持在膜上。
