Imamura T, Oka S, Tanahashi T, Okita Y
Cell Biology Laboratory, National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Exp Cell Res. 1994 Dec;215(2):363-72. doi: 10.1006/excr.1994.1353.
Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.
先前的研究表明，成纤维细胞生长因子（FGF）-1一级结构中功能性核定位序列的存在与其作为促有丝分裂原的活性相关，但与其诱导受体酪氨酸磷酸化的潜力无关，这表明FGF-1作为一种核蛋白存在尚未明确的功能。在本研究中，我们对外源性添加的FGF-1的胞质和核定位进行了研究。制备了FGF-1特异性单克隆抗体。通过广泛筛选，分离出了高特异性抗体克隆。对于BALB/c 3T3细胞和人脐静脉内皮（HUVE）细胞，用这些克隆进行的免疫荧光研究表明，在细胞周期的G1期，FGF-1从细胞质转运至细胞核。随后，在BALB/c 3T3细胞进入S期之前，FGF-1转移至核周和近核区域。共聚焦显微镜检查证实，核染色遍布整个核基质，在核膜边界和核仁处有一些富集。对经血清诱导增殖并在不同时间点用外源性FGF-1脉冲处理的BALB/c 3T3细胞进行分级分离后的免疫印迹分析显示，外源性FGF-1持续掺入细胞质，而在静止细胞受到刺激后5小时，核转位显著增加。通过向在FGF-1存在下培养的HUVE细胞显微注射抗FGF-1抗体表明，FGF-1的胞质形式存在于可溶性细胞质部分而非膜包裹的区室、内体中。数据表明，外源性添加的FGF-1持续被内吞并分级进入细胞质可溶性区室，而其核定位在核转位水平受到调控，且优选在细胞周期的G1晚期发生。
