Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid
Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid
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DOI：
10.1194/jlr.m100406-jlr200
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发表时间：
2002-10-01
影响因子：
6.5
通讯作者：
Lillehaug, JR
中科院分区：
文献类型：
--
作者：
Bjorndal, B;Helleland, C;Lillehaug, JR
当C3H/10TI/2细胞在含TTA的培养基中培养时，非β-氧化的十四烷基硫代乙酸（TTA）掺入细胞膜中。我们在这里证明，这种细胞膜的改变影响参与信号转导的蛋白质的核转位。细胞脂肪酸组成的分析表明，TTA和TTA：1n-8约占细胞/核膜中总脂肪酸的40mol%。在TTA处理的细胞中，c-fos表达的活化被显著抑制，但丝裂原活化蛋白激酶（ERK）的酶促活化不受影响。免疫荧光和共聚焦显微镜研究表明，在有丝分裂原刺激的TTA处理的细胞中，磷酸化ERK 1/2，蛋白激酶Calpha（PKCalpha）和PKC β（1）从细胞质到细胞核的易位显著减少和延迟。伴随着减少核输入，ERK 1/2去磷酸化减少在TTA处理的细胞。有没有TTA诱导的抑制核输入的蛋白质与经典的核定位信号（NLS），如所看到的BSA融合的NLS从SV40大T的体外核输入实验，或在体内研究的hnRNPA1核输入。输入蛋白α、输入蛋白β、输入蛋白7和NTF2的表达水平在TTA处理的细胞中没有改变。两者合计，我们的数据表明，TTA治疗引起细胞脂肪酸组成的变化，负面影响NLS独立的蛋白质易位通过核孔复合物的机制。
The non-p-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10TI/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase Calpha (PKCalpha), and PKCbeta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.