Truncated WT1 mutants alter the subnuclear localization of the wild-type protein
Truncated WT1 mutants alter the subnuclear localization of the wild-type protein
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DOI：
10.1073/pnas.92.26.11960
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发表时间：
1995-12-19
影响因子：
11.1
通讯作者：
Haber, DA
中科院分区：
文献类型：
--
作者：
Englert, C;Vidal, M;Haber, DA
WT 1编码一种锌指蛋白，表达为不同的亚型，在Wilms瘤的一个子集中失活。结构和体细胞突变破坏WT 1的DNA结合结构域，导致潜在的显性阴性表型。在产生表达野生型WT 1同种型和WT 1突变体的诱导型细胞系中，我们观察到诱导蛋白质的亚核定位的显著差异。以高亲和力结合至确定的DNA靶的WT 1同种型WT 1（-KTS）弥漫定位于整个细胞核。相反，具有降低的DNA结合亲和力的可变剪接变体WT 1（+KTS）的表达，或具有破坏的锌指结构域的WT 1突变体导致细胞核内的斑点状表达模式。虽然外观相似，但WT 1变体定位于亚核簇与必需剪接因子SC 35的定位明显不同，表明WT 1不直接参与前mRNA剪接，定位于亚核簇需要WT 1的N末端，截短的WT 1突变体和野生型WT 1（-KTS）的共表达导致它们的物理关联，WT 1（-KTS）从弥散型向斑点型的再分布，以及其反式激活活性的抑制。这些观察结果表明，不同的WT 1亚型和WT 1突变体具有不同的亚核区室，显性阴性WT 1蛋白在体内与野生型WT 1物理结合，并可能导致其在亚核结构内的隔离。
WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors, Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins, The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus, In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1(+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing, Localization to suhnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments, Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.