Taperin (C9orf75) is a nuclear PP1α binding protein expressed as multiple isoforms
We have previously shown the merits of i) SILAC-based quantitative proteomics on the interactome of GFP-PP1 immunoprecipitations (Trinkle-Mulcahy et al., 2006
; Trinkle-Mulcahy et al., 2008
) and ii) peptide displacement affinity chromatography (Moorhead et al., 2008
) for the unbiased identification of nuclear PP1 regulatory subunits. The latter method relies on the binding of microcystin sensitive phosphatases to a matrix coupled with the toxin and the specific release of PP1 associated proteins by incubation with excess peptide encompassing an RVXF motif, the canonical and primary PP1 interaction region within most regulatory subunits (Moorhead et al., 2007
). Initial data from the ensuing mass spectrometry defined nuclear PP1 interactomes that showed partial overlap between both approaches, with one common candidate being c9orf75/taperin (Moorhead et al., 2008
; Trinkle-Mulcahy et al., 2006
). Considering the recent identification of taperin as a protein mutated in patients with non-syndromic, hereditary deafness, we investigated this interaction in more detail.
Four human taperin isoforms, generated by alternative splicing events, have been described thus far. The canonical isoform 1 is 711 amino acids long (predicted mass of 75.6 kDa), while the remaining 3 all miss the initial 306 N-terminal amino acids with predicted masses of 44.1, 47.2 and 44.2 kDa, respectively (see supplementary material Fig. S1 for isoform sequence alignments). Isoform 1 is supported by its alignment with mouse sequences and transcript evidence, yet at the time of our study no actual protein based observations supported this. Sequence analysis further showed that all taperin isoforms possessed one potential PP1 docking motif (KISF) between amino acids 577–580 in isoform 1 and amino acids 271–274 in isoforms 2–4. We therefore cloned and expressed isoform 2 (44.2 kDa) as a His-tagged fusion protein for recombinant protein studies and polyclonal antibody production (supplementary material Fig. S2). Antibodies generated against this isoform should ensure recognition of all versions of taperin. The resulting, affinity purified antibodies decorated two major (~44 and 47kDa) and several minor bands in HeLa and U2OS extracts (supplementary material Fig. S3B, S4B). Further Western blot analyses of various rat tissues identified multiple immunoreactive bands, predominantly around 47 kDa but also a ~75 kDa version in testis (supplementary material Fig. S4A), a mass reminiscent of the predicted isoform 1 size, and several weaker signals of ~95 kDa. Interestingly, Western blot analyses mainly recognized ~44–47 kDa sized taperin isoforms, yet our mass spectrometry results derived from HeLa cells not only identified peptides shared between all four isoforms, but also peptides unique for isoform 1 (supplementary material Fig. S1), thereby providing support for the presence of isoform 1 in certain cells and/or tissues.
Next, we focused on the interaction between PP1 and taperin. We incubated nuclear HeLa extracts with a microcystin matrix to enrich phosphoprotein phosphatases and performed peptide displacement with a RARA peptide (scrambled PP1 interaction motif), a RVRW peptide (GKKRVRWADLE) and finally by sodium thiocyanate (SCN) to selectively elute PP1 binding proteins. Eluted proteins were separated by SDS-PAGE and probed with a polyclonal antibody against taperin (). Complete displacement with the RVRW peptide suggests association with PP1 occurs primarily through the PP1 dock site (KISF) within taperin. Interestingly, peptide-released taperin ran on SDS-PAGE at an apparent mass of ~95 kDa (), a size observed as a minor band in Western blotting (supplementary material Fig. S4). We corroborated these results by immunoprecipitation of endogenous taperin from HeLa cell extracts and revealed that taperin preferentially binds PP1α over PP1γ, but does not bind PP1β (). To exclude the possibility that PP1 associates with taperin through another protein, we used recombinant DIG-labeled PP1α and PP1γ to perform overlay (Far-Western) experiments with taperin. We again demonstrated a strong preference for recombinant taperin to bind PP1α over PP1γ () with the association being completely abolished if the putative PP1 dock site, KISF, is mutated to KASA. Finally, in vitro pulldowns with recombinant taperin and PP1α show a robust interaction, again abolished when the putative PP1 binding KISF motif is mutated (), establishing this as the primary PP1 dock site on taperin. These results are in line with our quantitative proteomics approach, and identify C9orf75/taperin as a nuclear PP1 interacting protein with a preference for PP1α over PP1γ (supplementary material Fig. S5).
Taperin (c9orf75) preferentially associates with PP1α.
Taperin inhibits PP1 activity
Using glycogen phosphorylase a
as a substrate, we found that taperin is a strong inhibitor of PP1α activity (). In support of an interaction between PP1 and taperin via its KISF sequence we were able to relieve the inhibition of PP1α activity by titrating in an RVRW peptide, whose high affinity for PP1 disrupted the association of PP1 with the KISF interaction site on taperin (Moorhead et al., 2008
). Titrating in a scrambled (RARA) peptide where the key PP1 interaction residues were mutated to alanine had no effect on PP1 activity supporting the idea of KISF as the primary PP1 binding region on taperin.
Taperin is a soluble and predominantely nucleoplasmic protein
Immunostaining of permeabilized, fixed HeLa cells with taperin antibodies revealed that taperin is a predominately nuclear protein that does not accumulate within nucleoli (). This was further confirmed by transiently expressing GFP-taperin in HeLa () and U2OS () followed by live cell imaging.
Taperin is predominantly nucleoplasmic in vivo.
Our SILAC-based proteomic experiments and Western blot analyses suggested that taperin could associate with PP1 in both nucleoplasm and cytoplasm (supplementary material Fig. S4B, S5) whereas immunofluorescence () suggests a clear enrichment of taperin in the nucleoplasm. We have occasionally observed this for other soluble, shuttling nuclear proteins, suggesting it may be a fractionation artifact. Alternatively, natural cytoplasmic re-localization may occur only under specific cellular conditions. The ability of taperin to shuttle between the nucleus and cytoplasm was demonstrated using both heterokaryon and fluorescence loss in photobleaching (FLIP) experiments (). In the heterokaryon approach, human cells expressing nucleoplasm accumulated GFP-taperin are fused to non-transfected mouse cells in the presence of cycloheximide to preclude expression of new GFP-taperin. DAPI staining reveals the origin of each nucleus (mouse nuclei show a characteristic pattern of large heterochromatin foci). We observed a fraction of GFP-taperin in the mouse nucleoplasm following fusion, indicating that it migrated from the human nuclei in the heterokaryon (). This observation was supported by FLIP experiments in which we repeatedly photobleached a region in the cytoplasm in U2OS cells expressing either GFP or GFP-taperin (). We followed its impact by measuring GFP-intensity of a nucleoplasmic area. The bulk of the nucleoplasmic GFP signal is lost from the cell after 35 bleach cycles, while a similar loss of the nucleoplasmic GFP-taperin signal requires 60 bleach cycles. In addition to confirming that taperin shuttles, it also establishes that it is not quite as dynamic as free GFP, an observation supported by FRAP experiments that showed a rapid, albeit slower than GFP, recovery rate for photobleached nucleoplasmic GFP-taperin (data not shown). Taken together, taperin can be regarded as a predominantly nucleoplasmic protein that has the ability to shuttle out of the nucleus.
Taperin can shuttle between the nucleus and cytosol.
In vivo interaction between taperin and PP1
The in vivo interaction of PP1 with taperin was confirmed using Bimolecular Fluorescence Complementation (BiFC) and co-transfecting U2OS cells with fragments of the EYFP protein fused to either PP1γ or PP1 targeting subunits, namely NIPP1, a well-established nuclear PP1 binding protein which serves as control, taperin or mutated taperin (data not shown). The direct vicinity (<10nm) of CYFP-PP1 constructs with either NYFP-NIPP1 or NYFP-taperin formed a competent fluorophore, emitting a clear nuclear EYFP signal (). Mutation of the taperin PP1 docking motif KISF to KASA completely abolished the signal (data not shown). The majority of the taperin-PP1 YFP signal is nucleoplasmic, yet a small fraction can be observed in the cytoplasm. This again suggests that taperin shuttles between these two cellular compartments.
Bimolecular fluorescence complementation (BiFC) demonstrates the in vivo interaction of taperin and PP1.
It has been shown that over-expression of PP1 regulatory subunits can cause a relocation of PP1 itself, which functions as a strong indicator of their in vivo
interaction (Trinkle-Mulcahy et al., 2001
). To support the BiFC experiments we studied PP1 relocalization in HeLa cells stably expressing low levels of either EGFP-PP1γ or EGFP-PP1α and transiently expressing either wild type (mCherry-taperin) or mutated (mCherry-taperinKASA
) taperin. As shown in , over-expression of taperin recruits most of the nuclear PP1γ, including the nucleolar pool (arrow), to the nucleoplasm of the cell where taperin resides. Mutation of the PP1 binding site from KISF to KASA abolishes this relocalization of PP1γ (). As PP1α is already nucleoplasmic, it is difficult to assess whether over-expressed taperin recruited additional PP1α to its localization site (). Over-expressed taperin remains complexed with PP1 during mitosis, as shown by the failure of EYFP-PP1γ to be recruited to metaphase kinetochores in the presence of excess ECFP-taperin (supplementary material Fig. S6A). Conversely, overexpression of the KASA mutant of taperin has no obvious effect on mitotic PP1γ localization (supplementary material Fig. S6B).
In vivo relocalization of PP1 by exogenously over-expressed taperin.
These data are further supported by heterokaryon experiments in which mCherry-taperin is introduced into cells to dynamically relocalize EGFP-PP1γ that has already been targeted to its normal intranuclear sites of action. In brief, HeLa cells transiently expressing mCherry-taperin are fused to EGFP-PP1γ expressing cells in the presence of cycloheximide to inhibit new protein translation. When mCherry-taperin enters the nuclei of cells already containing EGFP-PP1γ, it overrides the normal localization pattern by competing PP1 away from other nuclear targeting subunits, resulting in recruitment of the majority of nuclear PP1γ to the nucleoplasm (). This also highlights the high affinity of the taperin-PP1 interaction. Recruitment does not occur when the PP1 dock site in taperin is mutated to KASA, with PP1γ maintaining its normal nuclear distribution ().
Additional taperin interaction partners
To assess taperin interaction partners, we carried out a SILAC-based quantitative immunoprecipitation of taperin from nuclear extracts of HeLa cells transiently expressing ECFP-taperin. Mass spectrometry and quantitation of heavy: light amino acid ratios confirmed once again the co-precipitation of PP1 with taperin but also identified the classic DNA damage proteins Ku70, Ku80, PARP1, TOPOI and TOPOIIα (supplementary material Table S1). These were the only proteins identified with more than one peptide and ratios >1, suggesting they are bona fide
taperin interactors. To confirm this observation we transiently expressed either GFP alone or GFP-taperin in HeLa cells, immunoprecipitated both from whole cell extracts using the high affinity GFP-Trap®_A reagent and probed Western blots of the eluted complexes with antibodies specific to these putative interaction partners. As shown in , each of these proteins is indeed enriched with GFP-taperin compared to GFP alone. It should be noted that the weak Ku70/80 bands detected in the control IP are not surprising, as these abundant proteins are known “bead contaminants” that can bind non-specifically to affinity matrices (Trinkle-Mulcahy et al., 2008
Taperin associates with topoisomerases I and IIα, PARP and Ku.
Taperin accumulates at sites of DNA damage
Given that taperin appears to interact with several proteins known to play a role in the DNA damage response, we sought to determine if taperin is recruited to sites of DNA damage. Several other PP1 regulatory subunits have recently been identified as regulators of the DNA damage response, including GADD34, RepoMan and PNUTS (Kuntziger et al., 2011
). We used U2OS cells, transiently expressing GFP alone (as a negative control), PNUTS-GFP (as a positive control), GFP-taperin (WT) or GFP-taperinKASA
. Cells were pre-sensitized to double strand breaks by staining with Hoechst 33342 (see for experimental design) and DNA lesions were induced by UV laser micro-irradiation of discrete regions in the nucleus, similar to the work of Landsverk et al. (Landsverk et al., 2010
) with PNUTS-GFP. As expected, GFP alone shows no accumulation at damage foci (data not shown), whereas PNUTS-GFP readily accumulates at these sites (). GFP-taperin displays an even faster recruitment () to DNA damage foci than PNUTS-GFP () but not to the same signal intensity (). The PP1 docking mutant (KASA) of taperin showed very similar kinetics to the PP1 binding version (), suggesting that taperin recruitment may occur prior to and/or independent of PP1 binding. Interestingly, sequence analysis of taperin revealed 2 RGG motifs that may facilitate nucleic acid binding (supplementary material Fig. S7).
Taperin is recruited to sites of DNA damage in vivo.
Taperin is a vertebrate specific protein
The human taperin sequences (isoforms 1–3) were used as a query in a series of pBLAST and tBLASTn searches to identify and collect homologous sequences. Taperin sequences were only found in vertebrates, but within those, mammals, birds and fish are present with no amphibian or reptile sequences represented (supplementary material Fig. S8). Within the taperin sequences, highest conservation is found in the C-terminal half where the PP1 docking motif (KISF) lies in a highly conserved stretch.
Relationship to phostensin
Database searching with the taperin primary sequence revealed phostensin, a previously characterized PP1 interacting protein, as the only sequence with homology with taperin. In turn, we used phostensin sequences as bait in a series of pBLAST and tBLASTn queries, which identified taperin and phostensin homologues. Once again, these are only found in vertebrates with examples in placental and marsupial mammals, but not in monotremes.
Direct alignment of phostensin and taperin reveals some degree of similarity along their entire length (supplementary material Fig. S7). Only a short domain in phostensin is highly related to taperin, and this contains the recognized PP1 interaction region of both proteins. The KISF PP1 dock site is a less frequent, functional version of the common RVXF motif and is found in both taperin and phostensin, with the residues bracketing this site also being well conserved. The sequences surrounding the PP1 docking motif were highly conserved throughout all BLAST derived taperin and phostensin sequences. We therefore used this region to develop a phylogenetic tree, to deduce the evolutionary relationship between both proteins (supplementary material Fig. S8). Both taperin and phostensin sequences align with similar sequences throughout vertebrate organisms, suggesting that these proteins may have originated from a common ancestral protein early in vertebrate evolution.
Previously, taperin exon 1 has been proposed as a mutational hotspot, prone to internal re-arrangements. We suspected that such events could also be observed in the amino acid sequence. Furthermore, considering the relationship between taperin and phostensin, we questioned whether phostensin could possess similar properties. To test the hypothesis of internal re-arrangements throughout evolution for each protein and to assess similarity between both, we generated “HHBlits” to perform “self against self” and “taperin against phostensin” dotmatcher comparisons. HHBlits use P.s.i.-Blast to collect a series of related sequences, derives a Hidden Markov Model (HMM) for the query and its hits, and compares it to a pre-computed database of HMMs formed by sets of related database sequences. Dotmatcher analyses provide a visual representation of the similarity between 2 sequences. Dotplot analyses of self against self comparisons show that not only taperin but also phostensin possess internal rearrangements, exemplified by the offset distribution of short fragments from the diagonal (supplementary material Fig. S9). Moreover, a dotplot of phostensin (613 amino acids) against taperin (711 amino acid version; supplementary material Fig. S9) shows a main diagonal at the N- and C-termini, suggestive of direct similarity, but also several offset diagonals within the interior of the molecules. Thus, taperin and phostensin do share homology outside the highly conserved domain but most likely also have a complex history of differential internal rearrangement from an ancestral sequence to bring about the pattern of internal repeats now observable.
We previously identified phostensin (KIAA1949) in our SILAC-based cellular interactome screen as a PP1α interacting protein (Trinkle-Mulcahy et al., 2006
). Here we confirm this interaction in both cytoplasm and nucleoplasm PP1 interactome screens (supplementary material Fig. S5). Phostensin has been described as a 165 amino acid protein (Kao et al., 2007
), but recent, database-annotated versions predict the existence of multiple longer versions, up to 613 amino acids. Indeed, mass spectrometry data from our PP1α pulldown identified phostensin peptides spread across the entire predicted 613 amino acid protein (supplementary material Fig. S10). We expressed full-length phostensin as a GFP-tagged fusion protein in HeLa cells and saw an exclusively cytosolic localization pattern with a strong enrichment at the plasma membrane (supplementary material Fig. S11). We also noted significant co-localization with mCherry-actin at both the cell periphery and at stress fibers, which supports a suggested role for phostensin in regulation of actin dynamics (Lai et al., 2009
). The cytosolic localization of phostensin is in sharp contrast to taperin, which is a predominantly nucleoplasmic protein. Finally, co-expression of full-length ECFP-phostensin with either EYFP-PP1α or –γ does not cause obvious relocalization of the phosphatase, suggesting that the interaction with PP1 is weaker than that observed for taperin and PP1 (data not shown) or, association may rely on specific cellular conditions.