Brook Lemlem, Whitfield G Kerr, Hsieh David, Bither Ryan D, Hsieh Jui-Cheng
Department of Basic Medical Sciences, College of Medicine-Phoenix, University of Arizona, Phoenix, Arizona.
Mount Auburn Hospital, 330 Mt Auburn St, Cambridge, Massachusetts.
J Cell Biochem. 2017 Feb;118(2):341-350. doi: 10.1002/jcb.25641. Epub 2016 Sep 21.
The mammalian hairless (Hr) protein plays critical roles in skin and brain tissues, but how it interacts with DNA and partner protein is only now being defined. Our initial tests of four consensus response elements, revealed that rat Hr can specifically bind to a consensus p53 response element (p53RE), 5'-AGACATGCCTAGACATGCCT-3', but not to response elements for NF-κB, TCF4 or Sp1. We then employed ChIP assays which verified that human HR binds to a p53RE of the GADD45A gene in both HEK293 (embryonic kidney) and U87 (glioblastoma) cells. Further, HR was shown to interact directly with the p53 protein in a co-immunoprecipitation assay. Cotransfections with p53RE reporter gene constructs revealed that rat Hr can boost p53-mediated transactivation of a reporter gene linked to the GADD45A p53RE, but blunts p53-mediated transactivation when the reporter gene is linked to a p21 promoter fragment containing a p53RE, with implications for the regulation of these two cell cycle control genes. Finally, our investigations of HR phosphorylation revealed that rat Hr is a substrate for PKC, but not PKA, and that human HR is phosphorylated in intact U87 cells at Ser-416, located in a highly conserved region which partially fulfills the criteria of a PKC site. We propose that mammalian Hr is a phosphoprotein which can exert cross-talk with the p53 pathway with important implications for the regulation of cell proliferation and differentiation in tissues such as skin and brain where Hr is highly expressed. J. Cell. Biochem. 118: 341-350, 2017. © 2016 Wiley Periodicals, Inc.
哺乳动物无毛（Hr）蛋白在皮肤和脑组织中发挥着关键作用，但它如何与DNA及伴侣蛋白相互作用，目前才刚刚开始明确。我们对四个共有反应元件进行的初步测试表明，大鼠Hr能够特异性结合共有p53反应元件（p53RE），即5'-AGACATGCCTAGACATGCCT-3'，但不能结合NF-κB、TCF4或Sp1的反应元件。随后，我们采用染色质免疫沉淀（ChIP）分析方法，证实人HR在HEK293（胚胎肾）细胞和U87（胶质母细胞瘤）细胞中均能结合GADD45A基因的p53RE。此外，在共免疫沉淀分析中显示HR可直接与p53蛋白相互作用。用p53RE报告基因构建体进行共转染实验表明，大鼠Hr能够增强与GADD45A p53RE相连的报告基因的p53介导的反式激活作用，但当报告基因与含有p53RE的p21启动子片段相连时，大鼠Hr会减弱p53介导的反式激活作用，这对这两个细胞周期调控基因的调节具有重要意义。最后，我们对HR磷酸化的研究表明，大鼠Hr是蛋白激酶C（PKC）而非蛋白激酶A（PKA）的底物，并且人HR在完整的U87细胞中位于Ser-416处被磷酸化，该位点位于一个高度保守的区域，部分满足PKC位点的标准。我们提出，哺乳动物Hr是一种磷蛋白，它可与p53信号通路发生相互作用，这对皮肤和脑等高表达Hr的组织中细胞增殖和分化的调控具有重要意义。《细胞生物化学杂志》2017年第118卷：341 - 350页。© 2016威利期刊公司
