Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells.
Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells.
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将外部添加的 FGF-1 易位到细胞中，需要两个核定位信号从细胞质转运到细胞核。
DOI：
10.1021/bi047403m
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
S. Olsnes
中科院分区：
文献类型：
--
作者：
J. Wesche;J. Małecki;A. Wiedlocha;M. Ehsani;E. Marcinkowska;T. Nilsen;S. Olsnes
外部添加的 FGF-1 被转运到细胞核中。早期研究表明，FGF-1 含有 N 末端核定位信号 (NLS)，与刺激 DNA 合成有关。我们在这里提供的证据表明 FGF-1 包含第二个假定的 NLS (NLS2)，它位于 C 末端附近。它是一个二分 NLS，由两个由 10 个氨基酸组成的间隔区隔开的赖氨酸簇组成。 GFP 和二分 NLS 的融合蛋白比单独的 GFP 更有效地转运到细胞核中，表明它可以在活细胞中充当 NLS。 FGF-1 在 N 端 NLS (NLS1) 或二分 NLS2 的第一个簇中发生突变，与肝素和 FGF 受体结合，并与野生型生长因子类似地激活下游信号传导。 NLS2 第二簇的突变导致与肝素的相互作用受损并降低稳定性。当将带有突变 NLS1 或 NLS2 第一个赖氨酸簇的放射性标记 FGF-1 添加到 NIH/3T3 细胞中时，它被易位到胞浆中，但不能有效转运到细胞核。 FGF-1 的磷酸化通常发生在细胞核中，而野生型 FGF-1 在添加到细胞后会被磷酸化，而 NLS 突变体则不会。因此，看来 NLS1 和 NLS2 对于 FGF-1 向细胞核的有效转运都很重要。两个 NLS 中发生突变的 FGF-1 对 DNA 合成的刺激显着减少，表明向细胞核的有效转运可能与 DNA 合成的刺激有关。
Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.