Lamin A/C binding protein LAP2α is required for nuclear anchorage of retinoblastoma protein
Lamin A/C binding protein LAP2α is required for nuclear anchorage of retinoblastoma protein
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DOI：
10.1091/mbc.e02-07-0450
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发表时间：
2002-12-01
影响因子：
3.3
通讯作者：
Hutchison, CJ
中科院分区：
文献类型：
--
作者：
Markiewicz, E;Dechat, T;Hutchison, CJ
视网膜母细胞瘤的磷酸化依赖锚定。细胞核中的Rb蛋白对其功能至关重要。我们表明，它的口袋C结构域是必要的和足够的核锚定通过瞬时表达绿色荧光蛋白（GFP）嵌合体的Rb片段在组织培养细胞和提取的细胞与低渗溶液。使用Rb口袋A、B和C的谷胱甘肽S-转移酶融合的固相结合测定揭示了核纤层蛋白C专门与口袋C的直接结合。核纤层蛋白A/C的结合配偶体核纤层相关多肽（lamina-associated polypeptide，LN）2 α与囊袋C强结合而与囊袋B弱结合。当从可溶性核组分中免疫沉淀LAP 2 α时，核纤层蛋白A/C和低磷酸化Rb有效地共沉淀。类似地，通过使用抗GFP抗体共沉淀表达的GFP-Rb片段的免疫沉淀可共沉淀LAP 2 α，条件是口袋C存在于GFP嵌合体中。通过在组织培养细胞中过表达显性负核纤层蛋白突变体，内源性核纤层蛋白A/C和LAP 2 α重新分配到核聚集体中，Rb也被隔离到这些聚集体中。在原代皮肤成纤维细胞中，LAP 2 α以生长依赖性方式表达。在没有LAP 2 α的情况下，低磷酸化Rb在细胞核中的锚定明显减弱。总之，这些数据表明，低磷酸化Rb通过口袋C与LAP 2 α-核纤层蛋白A/C复合物的相互作用锚定在细胞核中。
The phosphorylation-dependent anchorage of retinoblastoma. protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathione S-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2alpha, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2alpha was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2alpha, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2alpha into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2alpha is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2alpha. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2alpha-lamin A/C complexes.