BAG3 蛋白Ser187磷酸化位点定点突变促进FRO细胞迁移和侵袭

Bcl-2相关抗凋亡蛋白3（Bcl-2 associated athanogene 3，BAG3）是BAG家族的重要成员，调节肿瘤细胞的黏附、迁移和侵袭，促进恶性肿瘤的复发和转移。我们的前期工作证明，PKCδ可催化BAG3的Ser187位点磷酸化。本文研究BAG3蛋白磷酸化修饰对甲状腺癌FRO 细胞迁移、侵袭的影响及其可能机制。通过定点突变的方法，将BAG3蛋白的187位丝氨酸突变为天冬氨酸（S187D）模拟磷酸化，或者将丝氨酸突变为丙氨酸（S187A）抵抗磷酸化，从而间接推测BAG3蛋白Ser187位点磷酸化对FRO细胞迁移、侵袭的影响。 FRO细胞转染野生型BAG3、模拟磷酸化型BAG3、阻碍磷酸化型BAG3，通过划痕愈合实验和Transwell转移小室实验，观察BAG3蛋白磷酸化对FRO细胞迁移、侵袭的影响。进一步通过PKC激活剂和抑制剂，研究BAG3蛋白磷酸化对FRO细胞迁移、侵袭影响的机制。 结果显示，FRO BAG3-S187D模拟磷酸化组细胞在培养24 h、48 h时，划痕愈合率分别达到35%和80%。Transwell及三维Matrigel转移小室实验显示，平均每个视野穿膜细胞数分别达到180和350个，与对照组相比，差异有统计学意义（P<0.05）。PKC激活剂TPA及抑制剂Rottelerin处理FRO WT-BAG3细胞，24 h愈合率分别为40% 和15%，48 h划痕愈合率分别为55%和18%，Transwell穿膜细胞数分别为240和70个，与对照组细胞相比，差异显著（P<0.05）。本研究提示，BAG3蛋白Ser187磷酸化修饰，可促进甲状腺癌FRO细胞迁移、侵袭，其机制可能与PKC信号通路有关。

BAG3 Ser187 Loci Mutation Promotes Migration and Invasion of FRO Cells

Bcl-2 associated athanogene 3 (BAG3）is one of the important members of the BAG family. BAG3 regulates the adhesion, migration and invasion of tumor cells, and promotes the recurrence and metastasis of malignant tumor. Our preliminary work had proved that PKCδ can phosphorylate BAG3 at Ser187. In this paper, we studied the effect of BAG3 phosphorylation on migration and invasion in thyroid cancer FRO cells. The serine at BAG3 187 loci was mutated to aspartic acid or alanine, and then the influence of phosphorylation on migration and invasion of FRO cells was studied. FRO cells were stably transfected with WT-BAG3, BAG3-S187D or BAG3 S187A, then wound-healing and transwell assays were used to investigate the impact of BAG3 on migration and invasion potency of FRO cells. Furthermore, the effects of PKC protein inhibitor or activator were studied. The results indicated that the rates of wound healing nearly came to 35% and 80% after 24 hours or 48 hours in FRO BAG3-S187D group, and the average invaded cells came to 180 and 350 for transwell migration and invasion assays, in which the difference was statistically significant as compared with control group (P<0.05). FRO BAG3-WT cells treated with TPA or Rottelerin, the rates of wound healing nearly came to 40% and 15% after 24 hours, and the wound healing rates came to 55% and 18% after 48 hours. The average of invaded cells came to 240 and 70 with the statistically significance as compared with control group (P<0.05). Our results indicated that the phosphorylation of BAG3 at Ser187 induced migration and invasion of FRO cells, which may be related with PKC signal pathway.