埃兹蛋白477位酪氨酸的磷酸化在神经生长因子前体促进乳腺癌细胞侵袭中起关键作用

目的研究神经生长因子前体（precursor of nerve growth factor, proNGF）促进乳腺癌细胞侵袭的作用与埃兹蛋白（ezrin） 表达水平及其567位苏氨酸（Thr567）和477位酪氨酸（Tyr477）的磷酸化的相互关系。方法用梯度浓度的proNGF（0、2.5、5和 10 ng/mL）刺激人乳腺癌细胞系MDA-MB-231 和MCF-7，用Transwell 侵袭实验检测proNGF对MDA-MB-231 和MCF-7 侵袭 的影响；用免疫印迹法检测ezrin 蛋白的表达水平，ezrin Thr567 和ezrin Tyr477 磷酸化水平的变化；在MDA- MB-231 中转染 pEnter-His-ezrinY477F质粒（ezrin显性负突变质粒），研究ezrin Tyr477磷酸化在proNGF促进乳腺癌细胞侵袭中的作用。结果 proNGF 以浓度依赖的方式促进MDA-MB-231 和MCF-7 的侵袭（P<0.05）；proNGF 以浓度依赖和时间依赖的方式显著升高 ezrin Tyr477磷酸化，而对ezrin蛋白的表达以及其Thr567磷酸化无明显影响；Src激酶特异抑制剂SKI-606显著抑制proNGF对 ezrin Tyr477磷酸化的促进作用；转染pEnter-His-ezrinY477F抑制proNGF对MDA-MB-231细胞ezrin Tyr477磷酸化和侵袭能 力的促进作用（P<0.05）。结论ezrin Tyr477磷酸化在proNGF促进乳腺癌细胞侵袭中起关键作用；proNGF通过激活Src激酶使 ezrin Tyr477发生磷酸化。

Phosphorylation of ezrin Tyr477 is critical in invasion of breast cancer cells stimulated by precursor of nerve growth factor

Objective To investigate the effect of precursor of nerve growth factor (proNGF) in promoting invasion of breast cancer cells and its relation with ezrin expression and phosphorylation of ezrin Thr567 and Tyr477. Methods Human breast cancer cell lines MDA-MB-231 and MCF-7 were stimulated by gradient concentrations of proNGF (0, 2.5, 5 and 10 ng/mL) for 16 h, and the invasion of the cells was assessed with Transwell assay. The expression of ezrin and the phosphorylation of ezrin Thr567 and ezrin Tyr477 in the treated cells were examined by Western blotting. MDA-MB-231 cells were transfected with pEnter-His-ezrinY477F (a dominant negative mutant) to study the role of phosphrylation of ezrin Tyr477 in the invasion of breast cancer cell stimulated by proNGF. Results proNGF significantly promoted MDA-MB-231 and MCF-7 cell invasion in a concentration-dependent manner (P<0.05), and concentration- and time-dependently increased the phosphorylation of ezrin Tyr477 (P<0.05) without affecting the expression of ezrin or the phosphorylation of ezrin Thr567. The specific inhibitor of src, SKI-606, significantly inhibited the phosphorylation of ezrin Tyr477 induced by proNGF. Transfection with pEnter-His-ezrinY477F inhibited proNGF-induced invasion and phosphorylation of ezrin Tyr477 in MDA-MB-231 cells (P<0.05). Conclusion Phosphorylation of ezrin Tyr477 plays a critical role in the invasion of breast cancer cells stimulated by proNGF via proNGF/src/ezrin Tyr477 pathway.