Klein Sabrina, Meng Rui, Montenarh Mathias, Götz Claudia
Medical Biochemistry and Molecular Biology, Saarland University, 66424 Homburg, Germany.
Cancer Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 156 Wujiadun, Hankou, Wuhan 430045, China.
Pharmaceuticals (Basel). 2016 Dec 28;10(1):2. doi: 10.3390/ph10010002.
The homeodomain protein PDX-1 is a critical regulator of pancreatic development and insulin production in pancreatic β-cells. We have recently shown that PDX-1 is a substrate of protein kinase CK2; a multifunctional protein kinase which is implicated in the regulation of various cellular aspects, such as differentiation, proliferation, and survival. The CK2 phosphorylation site of PDX-1 is located within the binding region of the E3 ubiquitin ligase adaptor protein PCIF1. To study the interaction between PDX-1 and PCIF1 we used immunofluorescence analysis, co-immunoprecipitation, GST-pull-down studies, and proximity ligation assay (PLA). For the analysis of the stability of PDX-1 we performed a cycloheximide chase. We used PDX-1 in its wild-type form as well as phosphomutants of the CK2 phosphorylation site. In pancreatic β-cells PDX-1 binds to PCIF1. The phosphorylation of PDX-1 by CK2 increases the ratio of PCIF1 bound to PDX-1. The stability of PDX-1 is extended in the absence of CK2 phosphorylation. Our results identified protein kinase CK2 as new important modulator of the stability of PDX-1.
同源结构域蛋白PDX-1是胰腺发育和胰腺β细胞中胰岛素生成的关键调节因子。我们最近发现PDX-1是蛋白激酶CK2的底物；蛋白激酶CK2是一种多功能蛋白激酶，参与调节细胞的各个方面，如分化、增殖和存活。PDX-1的CK2磷酸化位点位于E3泛素连接酶衔接蛋白PCIF1的结合区域内。为了研究PDX-1与PCIF1之间的相互作用，我们采用了免疫荧光分析、免疫共沉淀、GST下拉实验和邻近连接分析（PLA）。为了分析PDX-1的稳定性，我们进行了放线菌酮追踪实验。我们使用野生型形式的PDX-1以及CK2磷酸化位点的磷酸化突变体。在胰腺β细胞中，PDX-1与PCIF1结合。CK2对PDX-1的磷酸化增加了与PDX-1结合的PCIF1的比例。在没有CK2磷酸化的情况下，PDX-1的稳定性得以延长。我们的研究结果确定蛋白激酶CK2是PDX-1稳定性的新的重要调节因子。
