Zhang Siyang, Guo Dawei, Jiang Lili, Zhang Qingfu, Qiu Xueshan, Wang Enhua
Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, PR China.
BMC Cancer. 2008 May 28;8:150. doi: 10.1186/1471-2407-8-150.
Suppressor of cytokine signaling 3 (SOCS3) is considered to inhibit cytokine responses and play a negative role in migration of various cells. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor kinase and has been found crucial to cell motility. However, little is known about whether SOCS3 could regulate PYK2 pro-migratory function in lung cancer.
The methylation status of SOCS3 was investigated in HBE and A549 cell lines by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine or transfected with three SOCS3 mutants with various functional domains deleted. Besides, cells were pretreated with a proteasome inhibitor beta-lactacystin where indicated. The effects of SOCS3 up-regulation on PYK2 expression, PYK2 and ERK1/2 phosphorylations were assessed by western blot using indicated antibodies. RT-PCR was used to estimate PYK2 mRNA levels. Transwell experiments were performed to evaluate cell migration.
SOCS3 expression was found impaired in A549 cells and higher PYK2 activity was correlated with enhanced cell migration. We identified that SOCS3 was aberrantly methylated in the exon 2, and 5-aza-2'-deoxycytidine restored SOCS3 expression. Reactivation of SOCS3 attenuated PYK2 expression and phosphorylation, cell migration was inhibited as well. Transfection studies indicated that exogenous SOCS3 interacted with PYK2, and both the Src homology 2 (SH2) and the kinase inhibitory region (KIR) domains of SOCS3 contributed to PYK2 binding. Furthermore, SOCS3 was found to inhibit PYK2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of PYK2 in a SOCS-box-dependent manner and interfered with PYK2-related signaling events, such as cell migration.
These data indicate that SOCS3 negatively regulates cell motility and decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells. These results also suggest a negative role of SOCS3 in PYK2 signaling, and a previously unidentified regulatory mechanism for PYK2 function.
细胞因子信号转导抑制因子3（SOCS3）被认为可抑制细胞因子反应，并在各种细胞迁移中发挥负向作用。富含脯氨酸的酪氨酸激酶2（PYK2）是一种非受体激酶，已发现其对细胞运动至关重要。然而，关于SOCS3是否能调节肺癌中PYK2的促迁移功能，人们所知甚少。
通过甲基化特异性PCR研究HBE和A549细胞系中SOCS3的甲基化状态。A549细胞用去甲基化剂5-氮杂-2'-脱氧胞苷处理，或用缺失不同功能域的三种SOCS3突变体转染。此外，在适当情况下，细胞用蛋白酶体抑制剂β-硫代乳酸预处理。使用指定抗体通过蛋白质印迹法评估SOCS3上调对PYK2表达、PYK2和ERK1/2磷酸化的影响。RT-PCR用于估计PYK2 mRNA水平。进行Transwell实验以评估细胞迁移。
发现A549细胞中SOCS3表达受损，较高的PYK2活性与增强的细胞迁移相关。我们确定SOCS3在第2外显子中发生异常甲基化，5-氮杂-2'-脱氧胞苷可恢复SOCS3表达。SOCS3的重新激活减弱了PYK2表达和磷酸化，细胞迁移也受到抑制。转染研究表明，外源性SOCS3与PYK2相互作用，SOCS3的Src同源2（SH2）结构域和激酶抑制区域（KIR）结构域均有助于与PYK2结合。此外，发现SOCS3可抑制A549细胞中与PYK2相关的ERK1/2活性。SOCS3可能以SOCS框依赖的方式促进PYK2降解，并干扰与PYK相关的信号事件，如细胞迁移。
这些数据表明，SOCS3负向调节细胞运动，甲基化诱导的SOCS3减少可能赋予A549细胞迁移优势。这些结果还提示了SOCS3在PYK2信号传导中的负向作用，以及一种先前未被识别的PYK2功能调节机制。
