14-3-3 zeta蛋白的胺基酸Y178對於其與Src交互作用及調控肺腺癌細胞侵襲的重要性

Abstract

Lung cancer, predominantly non-small-cell lung cancer (NSCLC), is the most common cause of cancer deaths worldwide, and metastasis is the major cause leading to mortality for cancer patients. To discover the novel metastasis- related genes, we used CGH array and identified a potential candidate, 14-3-3zeta, which plays a critical role in a wide variety of cellular processes and tumor progression. Moreover, our previous study indicated that 14-3-3zeta can induce EMT (epithelial-mesenchymal transition) to cause lung cancer metastasis; however, the underlying molecular mechanism is still unclear. Therefore, the objective of this study is to investigate the molecular mechanism of 14-3-3zeta protein in promotion of lung cancer invasion. First, we observed that overexpression of 14-3-3zeta enhanced cell invasive capability and MMP2 secretion from the cells. Meanwhile, 14-3-3zeta expression also facilitated the formation of podosome rings. Src is a major player in the induction of tumor invasion and its activity is increased in highly metastatic lung cancer cells. And Src is also known to regulate the formation of podosome ring. By using computer prediction, we found that 14-3-3zeta has the potential to interact with Src, and the interaction was further confirmed by co-immunoprecipitation and GST pull-down assay. In addition, we performed site-direct mutagenesis to generate the various 14-3-3zeta mutants. The results showed that Y178 mutant diminished the tyrosine-phosphorylation of 14-3-3zeta and accompanied with a decrease of the interaction between 14-3-3zeta and Src. Nevertheless, there was no difference in the p162/165 mutant. Owing to the Y178 in 14-3-3zeta is crucial for the binding of Src-SH2 domain, we introduced the various 14-3-3zeta constructs to lung cancer cells and demonstrated that Y178 phosphorylation is important for 14-3-3zeta protein to promote cell invasion, migration, and colony formation activity. We also noticed that Y178 mutant could rescue the alteration of epithelial plasticity, F-actin rearrangement, formation of F-actin ring and MMP2 secretion from the cells induced by wild type. Taken together, we presume that 14-3-3zeta may be involved in Src-mediated signaling, and suggest that the association of 14-3-3zeta with Src is important for cell invasion.