Klingenberg O, Wiedocha A, Citores L, Olsnes S
Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
J Biol Chem. 2000 Apr 21;275(16):11972-80. doi: 10.1074/jbc.275.16.11972.
Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.
酸性成纤维细胞生长因子（aFGF）是许多细胞的强效促有丝分裂原。外源性aFGF能够进入敏感细胞的细胞质和细胞核。有迹象表明，受体酪氨酸激酶的激活以及aFGF向细胞核的转位对于有丝分裂的发生都很重要。然而，aFGF从细胞表面运输到细胞核的机制尚不清楚。在这项研究中，我们证明，化学抑制剂以及PI 3-激酶显性负突变体的表达对磷脂酰肌醇（PI）3-激酶的抑制作用会阻止aFGF向细胞质和细胞核的转位。通过细胞分级分离、对修饰为含有法尼基化信号的aFGF进行法尼基化，以及对添加到细胞外部的aFGF进行蛋白激酶C磷酸化来监测向细胞质和细胞核的转位。如果aFGF与白喉毒素A片段融合，它可以通过白喉毒素途径从细胞表面人工转位到细胞质中。经过进一步孵育后，由于aFGF中的核定位序列，融合蛋白进入细胞核。我们在此证明，在抑制PI 3-激酶后，融合蛋白保留在细胞质中。我们还提供证据表明，融合蛋白的磷酸化状态并不调节其核质分布。
