Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein

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.

Keywords: DNA damage; Nonsyndromic deafness; Nucleus; Phostensin; Protein phosphatase one (PP1); Protein phosphorylation.

Fig. 1.. Taperin (c9orf75) preferentially associates with PP1α.

(A) HeLa cell nuclear extract was incubated with microcystin-Sepharose matrix, washed extensively and sequentially eluted with first the RARA peptide, followed by the RVRW peptide to specifically elute PP1 binding complexes, and finally a 3 M NaSCN step to elute any remaining protein. Eluates were then subjected to SDS-PAGE, transferred to nitrocellulose and a Western blot performed using affinity purified taperin antibodies (2 µg/mL). Endogenous taperin was immunoprecipitated (B) from a HeLa cell extract and blotted with the PP1 isoform specific antibody shown on the right. Control immunoprecipitation was with an equivalent amount of pre-immune IgG. Antibody specificity is shown by running 10 ng, 10 ng and 2 ng of recombinant purified PP1α, β and γ respectively, and as shown in supplementary material Fig. S12 and S13. (C) for far-Western blots, proteins were subjected to SDS-PAGE, transferred to nitrocellulose and overlaid with recombinant digoxigenin-coupled PP1α or PP1γ. Proteins are wild type [KISF] or mutated [KASA] 6His-taperin and 30 µg of rat nuclear extract as control. To ensure equal loading membranes were stripped and re-probed with anti-taperin (lower panels). For pulldowns (D), recombinant PP1α was mixed with Ni-NTA beads alone (control) or with recombinant wild type (KISF) or mutated (KASA) 6His-taperin. After release from the beads with SDS sample buffer, proteins were subjected to Western blot analysis with anti-taperin or anti-PP1 antibodies, as indicated. (E) the protein phosphatase activity of PP1α was monitored when mixed with an equal molar amount of taperin in the presence of increasing amounts of either RVRW peptide (—□—; GKKRVRWADLE) or RARA peptide (—♦—; GKKRARAADLE). Protein phosphatase activity was measured using phosphorylase a as the substrate. Data points are mean ± SD for n = 3.

Fig. 2.. Taperin is predominantly nucleoplasmic in vivo.

(A) Affinity purified taperin antibodies (5 µg/mL) were used to localize endogenous protein and DNA was stained with Hoechst in PFA-fixed HeLa cells (top panel), revealing a predominant nucleoplasmic localization in interphase and diffuse staining with no apparent accumulations in metaphase (arrow). Taperin is also predominantly nucleoplasmic when transiently expressed as a GFP-tagged fusion protein in both HeLa (B) and U2OS (C) cells. In these two panels the GFP images are shown superimposed on differential interference contrast (DIC) images to the right. Scale bars are 5 µM. Antibody controls are shown in supplementary material Fig. S3.

Fig. 3.. Taperin can shuttle between the nucleus and cytosol.

(A) In the heterokaryon approach depicted in the diagram, HeLa cells transiently expressing GFP-taperin (green) were mixed with non-transfected SW3T3 mouse cells. Cytoplasmic membranes were then fused by treatment with 50% PEG for 90 seconds and allowed to recover for 3 hours in media containing cycloheximide. DNA was then stained with Hoechst 33342 (blue) and cells imaged live to assess distribution of GFP-taperin between the original HeLa (arrowhead) and mouse (arrow) nuclei. The dashed line indicates the cell membrane. Scale bars are 5 µM. (B) Diagram depicting the FLIP experiment carried out to measure inter-compartmental dynamics of GFP-taperin, in which a cytoplasmic pool of the fusion protein is repeatedly bleached while the intensity of the nucleoplasmic pool is monitored over time. (C) Graph demonstrating the rapid loss of nucleoplasmic GFP (blue diamonds) when a cytoplasmic pool is bleached. The % maximum fluorescence intensity (normalized for photobleaching due to acquisition) is plotted versus the number of bleach events (each 100% laser power for a 0.05 sec duration). GFP-taperin (red squares) shows slower dynamics than free GFP, but a significant fraction of the nucleoplasmic pool is eventually bleached, indicating shuttling to the cytoplasm. (D) Sample dataset for each FLIP experiment. The lightning bolt indicates the photobleached region while the hashed circle shows the region of interest (ROI) that was monitored within the nucleus in each cell. To normalize for photobleaching caused by acquisition, a ROI was monitored in a neighboring cell. The number of bleach events is indicated in the bottom right corner. Each experiment was repeated 3 times. Scale bars are 5 µM.

Fig. 4.. Bimolecular fluorescence complementation (BiFC) demonstrates the in vivo interaction of taperin and PP1.

The diagram depicts the design of the BiFC experiment, in which PP1γ was fused to an 84 amino acid C-terminal fragment of EYFP (pC/EYFP-PP1) and a targeting subunit fused to a 154 amino acid N-terminal fragment of EYFP (pN-EYFP-targeting subunit). If the proteins interact directly the fragments will complement each other to form a fluorescent EYFP molecule that can be excited with 513 nm light to emit light at 528 nm. As a positive control, U2OS cells were transfected with pNYFP-NIPP1 and pCYFP-PP1γ to demonstrate the nucleoplasmic interaction of these two proteins (green). When pNYFP-taperin was co-transfected with CYFP-PP1γ, a signal was detected throughout the cell (green), indicating interaction of the two proteins in both the nucleus and the cytoplasm. DNA is stained with Hoechst 33342 (blue). No signal was detected when NYFP-taperin^KASA was co-transfected with CYFP-PP1γ (data not shown). Scale bars are 5 µM.

Fig. 5.. In vivo relocalization of PP1 by exogenously over-expressed taperin.

(A) Transient overexpression of mCherry-tagged taperin (red) in HeLa^EGFP-PP1γ cells relocalizes PP1γ (green) from nucleoli (arrow) and other nuclear sites of accumulation to the characteristic nucleoplasmic localization pattern of taperin itself. (B) As PP1α (green) exhibits a similar nucleoplasmic localization pattern to that of taperin, overexpression of mCherry-taperin (red) in HeLa^EGFP-PP1α cells does not lead to an easily observable difference. (C) Over-expression of the non-PP1 binding mutant mCherry-taperin^KASA does not lead to a relocalization of PP1γ in interphase HeLa^EGFP-PP1γ cells. (D) Heterokaryon experiment in which HeLa cells transiently over-expressing mCherry-taperin (red) were fused to HeLa^EGFP-PP1γ cells (green) in the presence of cycloheximide and imaged live 3 hours post-fusion. DNA was stained with Hoechst 33342 (blue). The dashed line indicates the cell membrane. The arrowhead indicates a nucleus containing both fusion proteins, in which PP1 is relocalized out of nucleoli (arrow) by taperin. (E) A similar experiment was carried out with the non-PP1 binding mutant mCherry-taperin^KASA (red), which does not relocalize PP1 out of nucleoli (arrow) in nuclei containing both fusion proteins (arrowhead). Scale bars are 5 µM.

Fig. 6.. Taperin associates with topoisomerases I and IIα, PARP and Ku.

Putative taperin interaction partners identified in a quantitative proteomic screen (supplementary material Table S1) were validated by co-IP and Western blot analysis. Whole cell extracts were prepared from HeLa cells transiently over-expressing either GFP-taperin or GFP alone and subjected to pulldown using the high affinity GFP-Trap_A^®. Following wash steps all proteins were eluted from the beads, separated by SDS-PAGE along with input lanes from crude extracts, transferred to nitrocellulose and probed with commercial antibodies raised against Ku70, Ku80, PARP1, TOPOI and TOPOIIα, as indicated. Each protein migrated at its expected molecular mass. PARP1 runs at 116 kDa, but after cleavage is know to have two dominant degradation products of 89 and 24 kDa as shown here. The asterisk (*) indicates a longer exposure of the IP lanes in the Ku70 Western blot.

Fig. 7.. Taperin is recruited to sites of DNA damage in vivo.

(A) Design of the experiment used to assess recruitment to discrete DNA lesions induced by UV laser microirradiation in pre-sensitized (by staining with Hoechst 33342) live cells. Following irradiation of a specific region of interest (ROI) the fluorescence intensity of GFP was then monitored in this same ROI over time. (B) Time-lapse imaging of GFP-taperin demonstrating recruitment of the fusion protein to the site of irradiation (arrow) over a 150 sec period. A pre-irradiation image was taken for comparison, and the first post-irradiation image was collected 0.002 sec after the laser fired. (C) The non-PP1 binding mutant GFP-taperin^KASA was subjected to the same treatment and demonstrated similar kinetics of recruitment to sites of UV-induced DNA damage. (D) As a positive control, the recruitment of PNUTS-GFP to DNA lesions was monitored over the same time scale. (E) The average % maximum fluorescence intensity ± SE was plotted versus time for GFP-Taperin (•, n = 10), GFP-Taperin^KASA (×, n = 10) and PNUTS-GFP (▴, n = 5). Data were normalized for photobleaching due to acquisition. The dashed line indicates the original fluorescence intensity within the ROI prior to DNA damage. Scale bars are 5 µm.