Wang Chun-Yu, Wang Yi-Ting, Tzeng Der-Wan, Yang Jia-Ling
Molecular Carcinogenesis Laboratory, Institute of Biotechnology & Department of Life Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan.
Toxicol Appl Pharmacol. 2009 Mar 1;235(2):244-52. doi: 10.1016/j.taap.2008.12.007. Epub 2008 Dec 24.
Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC --> ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1(S338) and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Gö6976 or depleting PKCalpha using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCalpha, Ras-GTP, phospho-Raf-1(S338) and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCalpha activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCalpha activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCalpha and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.
醋酸铅（Pb）是一种可能的人类致癌物，可在细胞外信号调节激酶1和2（ERK1/2）上游激活蛋白激酶C（PKC）。然而，Pb激活PKC→ERK1/2是否涉及受体/非受体酪氨酸激酶和Ras信号转导器仍不清楚。在此，我们证明了Pb引发的一种在CL3人非小细胞肺腺癌细胞中传递ERK1/2信号的新机制。Pb诱导更高的Ras-GTP稳态水平对于增加磷酸化Raf-1（S338）和磷酸化ERK1/2至关重要。用传统的PKC抑制剂Gö6976预处理细胞或使用特异性小干扰RNA耗尽PKCalpha可阻断Pb诱导的Ras-GTP。Pb还激活细胞酪氨酸激酶。针对表皮生长因子受体（EGFR）的特异性药理抑制剂PD153035和针对Src家族酪氨酸激酶（SFK）的SU6656，但针对血小板衍生生长因子受体的AG1296不能抑制Pb诱导的酪氨酸激酶、PKCalpha、Ras-GTP、磷酸化Raf-1（S338）和磷酸化ERK1/2。此外，在细胞暴露于Pb 1-5分钟和5-30分钟期间，分别发生了EGFR多个自磷酸化位点和保守的SFK自磷酸化位点上酪氨酸的磷酸化。有趣的是，Pb激活EGFR需要内在激酶活性但不需要受体二聚化。抑制SFK或PKCalpha活性不影响EGFR磷酸化，而敲低EGFR可阻断Pb后SFK磷酸化和PKCalpha激活。总之，这些结果表明，对Pb的反应中EGFR的即时激活对于SFK和PKCalpha的激活以及随后的Ras-Raf-1-MKK1/2-ERK1/2信号级联反应是必不可少的。
