Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein
Authors: Mhairi Nimick
Veerle De Wever
Delphine Chamousset
Greg B G Moorhead
Jens Andersen
Tony Ferrar
Laura Trinkle-Mulcahy
Journal: Biology Open (2012)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: DNA damage; Nonsyndromic deafness; Nucleus; Phostensin; Protein phosphatase one (PP1)
Affiliations: Department of Biological Sciences, University of Calgary, 2500 University Dr, Calgary, Alberta, T2N 1N4, Canada, Department of Cellular & Molecular Biology and Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada, Department of Biochemistry & Molecular Biology, University of Southern Denmark, Odense, 55 DK 5230, Denmark

Abstract:  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. 

Retracted: No

PubPeer comments:
 Hi, I find the description of the determination of taperin interactome via SILAC a bit confusing and I would appreciate if someone could clarify this for me. In the description of the method the authors say:  For PP1, quantitative immunoprecipitations were performed using nuclear and cytoplasmic extracts from SILAC-media grown HeLa cell lines stably expressing low levels of GFP-PP1u03b1 or GFP-PP1u03b3 with the unmodified parental cell line as control and the GFP-Trapu00ae_A reagent (Chromotek) for affinity binding, as described in (Trinkle-Mulcahy et al., 2006; Trinkle-Mulcahy et al., 2008). To quantitatively define the taperin-interactome, a ECFP-taperin construct or, as a control, ECFP alone, were transiently expressed in Hela cells, with both cultures grown in SILAC media. Nuclear extracts were prepared and affinity purifications performed with a monoclonal anti-GFP antibody (Roche) covalently conjugated to Protein G Sepharose as described (Trinkle-Mulcahy et al., 2006). For direct comparison, affinity and control matrices were combined for protein elution and all subsequent steps, including separation by 1D SDS-PAGE (to reduce complexity) and identification of eluted proteins by mass spectrometry. Quantitation of the heavy:light arginines and lysines in each peptide yields a  H:L or SILAC Ratio , a measure for distinction between non-specific binding of the protein to the affinity matrix (ratio u223c 1:1) or specific enrichment (ratio 1) I am not sure where the authors are getting the H:L (SILAC) ratio from. Normally I would ve thought that they compare between the peptide signals they get from ECFP-taperin to ECFP alone expressing cells, however, they explicitly state that both cultures were grown in SILAC media. They do say earlier that the unmodified cell line served as control, however, it is stated in such a way that I would think that it refers to the PP1 interactome experiments. Moreover, they say  affinity and control matrices . Would these refer simply to lysates from ECFP-taperin and ECFP cells respectively? Or do the matrices refer to something else I am not aware of? As I am not experienced in this type of experiments I am having a hard time understanding this description and started wondering whether it s me failing to understand it or the authors describing it in a confusing way. ,