Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding.
Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding.
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发表时间：
2001-09
影响因子：
4
通讯作者：
D. A. Eberhard;L. Karns;Scott R Vandenberg;C. Creutz
中科院分区：
文献类型：
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作者：
D. A. Eberhard;L. Karns;Scott R Vandenberg;C. Creutz
本研究探讨了膜联蛋白II（AnxII）的核质分配控制机制。AnxII和它的配体，p11，通过免疫荧光定位到U1242 MG细胞的细胞质区室，与最小的AnxII或p11检测核内。类似地，GFP-AnxII和GFP-p11嵌合体定位于内源性蛋白。同样，GFP-AnxII（1-22）被排除在细胞核之外，而GFP-AnxII（23-338）和单独的GFP分布在整个细胞中。免疫沉淀和生化研究表明，GFP-AnxII没有形成异聚体复合物与内源性p11和AnxII。因此，AnxII N-尾是必需的，足以引起GFP融合蛋白的核排斥，但这不涉及p11结合。在AnxII 3-12区域发现了核输出信号共有序列。共有突变体GFP-AnxII（L10 A/L12 A）证实了这些残基是核排斥所必需的。GFP-AnxII（1-22）的核排斥是温度依赖性和可逆的，核输出抑制剂来普霉素B（Lm B）引起GFP-AnxII或过表达的AnxII单体在核中积累。因此，AnxII单体可以进入细胞核并积极输出。然而，LmB对U1242 MG细胞中AnxII/p11复合物的定位几乎没有影响，表明该复合物被隔离在细胞质中。相比之下，LmB处理的v-src转化的成纤维细胞引起内源性AnxII在细胞核中积累。过钒酸盐可加速LmB诱导的AnxII的核积累，而染料木素可抑制LmB诱导的AnxII的核积累，这表明磷酸化促进了AnxII的核进入。因此，核排斥的AnxII的结果从核输出的单体和螯合的AnxII/p11复合物，并可能通过磷酸化调节。
This study investigated mechanisms controlling the nuclear-cytoplasmic partitioning of annexin II (AnxII). AnxII and its ligand, p11, were localized by immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were distributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p11 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nuclear exclusion of the GFP fusion protein but this does not involve p11 binding. A nuclear export signal consensus sequence was found in the AnxII 3-12 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these residues are necessary for nuclear exclusion. The nuclear exclusion of GFP-AnxII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monomer to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus and is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequestered in the cytoplasm. By contrast, LmB treatment of v-src-transformed fibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of AnxII. Thus, nuclear exclusion of AnxII results from nuclear export of the monomer and sequestration of AnxII/p11 complex, and may be modulated by phosphorylation.