Department of Hygiene and Public Health I, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
Toxicol Appl Pharmacol. 2011 Mar 15;251(3):209-16. doi: 10.1016/j.taap.2010.12.015. Epub 2011 Jan 8.
We examined the effects of cadmium chloride (CdCl₂) exposure on the expression and phosphorylation status of members of the Fos family, components of the activator protein-1 transcription factor, in HK-2 human renal proximal tubular cells. Following the exposure to CdCl₂, the expression of c-fos, fosB, fra-1, and fra-2 increased markedly, with different magnitudes and time courses. The levels of Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) also increased in response to CdCl₂ exposure. Although the elevation of c-fos transcripts was transient, c-Fos protein levels increased progressively with lower electrophoretic mobility, suggesting stabilization of c-Fos through post-translational modifications. Consistently, we observed phosphorylation of c-Fos at Ser362 and Ser374 in HK-2 cells treated with CdCl₂. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)-including extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal kinase, and p38-increased after CdCl₂ exposure, whereas treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 suppressed the accumulation and phosphorylation of c-Fos. We mutated Ser362 to alanine (S362A), Ser374 to alanine (S374A), and both residues to alanines (S362A/S374A) to inhibit potential phosphorylation of c-Fos at these sites. S374A or double S362A/S374A mutations reduced c-Fos level markedly, but S362A mutation did not. On the other hand, S362A/S374A mutations induced a more pronounced reduction in c-Fos DNA-binding activity than S374A mutation. These results suggest that while Ser374 phosphorylation seems to play a role in c-Fos stabilization, phosphorylation at two C-terminal serine residues is required for the transcriptional activation of c-Fos in HK-2 cells treated with CdCl₂.
我们研究了氯化镉（CdCl₂）暴露对人肾近端小管细胞 HK-2 中激活蛋白-1 转录因子成员 Fos 家族成员的表达和磷酸化状态的影响。暴露于 CdCl₂后，c-fos、fosB、fra-1 和 fra-2 的表达明显增加，幅度和时间进程不同。Fos 家族蛋白（c-Fos、FosB、Fra-1 和 Fra-2）的水平也随着 CdCl₂暴露而增加。虽然 c-fos 转录物的升高是短暂的，但 c-Fos 蛋白水平随着较低的电泳迁移率逐渐增加，表明通过翻译后修饰稳定了 c-Fos。一致地，我们观察到 CdCl₂处理的 HK-2 细胞中 c-Fos 的 Ser362 和 Ser374 磷酸化。细胞外信号调节蛋白激酶（ERK）、c-Jun NH₂末端激酶和 p38 等丝裂原激活蛋白激酶（MAPKs）的磷酸化形式在 CdCl₂暴露后增加，而 MAPK/ERK 激酶抑制剂 U0126 和 p38 抑制剂 SB203580 的处理抑制了 c-Fos 的积累和磷酸化。我们将 Ser362 突变为丙氨酸（S362A）、Ser374 突变为丙氨酸（S374A）以及两个残基突变为丙氨酸（S362A/S374A）以抑制 c-Fos 在这些位点的潜在磷酸化。S374A 或双 S362A/S374A 突变显著降低了 c-Fos 水平，但 S362A 突变没有。另一方面，S362A/S374A 突变比 S374A 突变诱导了更明显的 c-Fos 转录激活活性降低。这些结果表明，虽然 Ser374 磷酸化似乎在 c-Fos 稳定化中起作用，但在 CdCl₂处理的 HK-2 细胞中，c-Fos 的转录激活需要两个 C 末端丝氨酸残基的磷酸化。
