14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells.

14-3-3蛋白（14-3-3 proteins）是一类调控性信号分子家族，可通过磷酸化依赖的方式与其他蛋白相互作用，并在信号转导通路中充当衔接蛋白或支架蛋白。家族成员14-3-3ζ（14-3-3ζ）被认为参与细胞信号传导、细胞周期调控及细胞凋亡过程。本实验室此前开展的系统性蛋白质组学分析，将信号转导与转录激活因子3（signal transducers and activators of transcription 3, Stat3）鉴定为一种新型14-3-3ζ相互作用蛋白。基于这一初始发现，本研究提供了14-3-3ζ与Stat3发生物理相互作用的实验证据。本研究进一步证实，Stat3的Ser727位点磷酸化对于14-3-3ζ的结合至关重要，而将Ser727突变为丙氨酸（Alanine）则会阻断14-3-3ζ与Stat3的结合。在U266细胞中抑制14-3-3ζ的蛋白表达，可降低Stat3的Ser727位点磷酸化水平并抑制其核转位，同时减弱Stat3的DNA结合活性与转录活性。此外，14-3-3ζ参与调控蛋白激酶C（protein kinase C, PKC）的活性，且14-3-3ζ与Stat3的结合可阻止Stat3的Ser727位点被蛋白磷酸酶2A（protein phosphatase 2A, PP2A）去磷酸化。综上，本研究结果支持如下模型：多条信号通路均可作用于Stat3，而14-3-3ζ作为关键协调因子，介导不同信号通路调控多发性骨髓瘤细胞中Stat3的激活与功能。