Department of Biological Sciences, University of Calgary , 2500 University Dr, Calgary, Alberta, T2N 1N4 , Canada.
Biol Open. 2012 Feb 15;1(2):128-39. doi: 10.1242/bio.2011049. Epub 2011 Nov 24.
The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic 'RVxF' motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.
蛋白磷酸酶 1(PP1)的杂乱无章的活性受到与其相关的蛋白的调控,这些蛋白被称为调节或靶向亚基。通过生化和蛋白质组学方法,我们证明常染色体隐性非综合征性听力损失基因 taperin(C9orf75)编码的蛋白优先将 PP1 的α同工酶对接。Taperin 通过经典的“RVxF”基序与 PP1 结合,并抑制酶的一般磷酸酶活性。Taperin 的稳态定位主要在核内,但我们在此证明该蛋白可以在核和细胞质之间穿梭,并且在这两个细胞区室中都发现其与 PP1 复合物化。尽管最初被鉴定为听力损失基因,但使用针对 taperin 的特异性抗体的 Western blot 分析表明,该蛋白在哺乳动物组织中广泛表达,存在多种剪接变体。Taperin 是脊椎动物谱系中出现的最近的蛋白质组新增物,其与 PP1 结合的位点嵌入在该蛋白最保守的区域内。Taperin 还与细胞质肌动蛋白结合蛋白 phostensin 具有祖先关系,后者也是与 PP1 相互作用的伴侣。采用基于定量稳定同位素标记的氨基酸培养(SILAC)的质谱法来揭示其他的 taperin 结合伴侣,并发现与 DNA 损伤反应蛋白 Ku70、Ku80、PARP 和拓扑异构酶 I 和 IIα 相互作用。与此一致,我们证明了 taperin 被主动募集到 DNA 损伤部位。这使得 taperin 成为参与 DNA 损伤修复途径的 PP1 靶向亚基家族的新成员。