Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif

PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-b family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a nove

PEDF interaction with Transportin-SR2 identified by Yeast two Hybrid screening and confirmed by <i>in vitro</i> interaction of recombinant proteins.

<p>pEG202PEDF^41–121 was transformed into EGY48 yeast strain, subsequently transformed with a foetal human brain library. Blue-white selection in presence of X-Gal identified possible interaction candidates. A. List of unique Yeast two Hybrid clones expressing PEDF^41–121 interaction candidates. B. Positive interactions identified by the screening. Clone H expressing LexA- PEDF^41–121 and HA-TRN-SR2, and EGY48 yeast transformed with pEG202 empty vector were grown on different selection media as indicated. C. Verification of bait and target fusion protein expression. pEG202PEDF^41–121 and pJG4-5-TRN-SR2^827–923 recovered from clone H were transformed into yeast; As controls, empty vectors pJG4-5 and pEG202 were also transformed into yeast. Yeast lysates were subjected to immunoblotting using a polyclonal anti-LexA and a polyclonal anti-HA antibody. D. Verification of PEDF and TRN-SR2 interaction by GST-pull down. GST, GST-TRN-SR2 full length and PEDF were cloned and purified according to the methods. GST or GST-TRN-SR2 were incubated with glutathione-agarose for 1 hr followed by addition of recombinant PEDF for 4 hr at 4°C. Samples were subjected to immunoblotting using a monoclonal GST, a monoclonal PEDF and monoclonal Transportin-SR2 antibody. E. Verification of PEDF and TRN-SR2 interaction by co-immunoprecipitation. Recombinant PEDF was incubated with GST or GST-TRN-SR2 for 4 h and monoclonal PEDF antibody pre-absorbed on Protein-A agarose for 1 hr at 4°C Samples were subjected to immunoblotting using a monoclonal anti-GST, a monoclonal anti-PEDF and monoclonal anti-Transportin-SR2. F. RT-PCR detection of TRN-SR2 expression in HEK293 cells used for transfections in this study, and in retinal pigment epithelial cells and HUVEC cells known to contain nuclear PEDF.</p

Mutation of the YxxYRVRS motif blocks nuclear import of PEDF.

<p>HEK293T cells were transiently transfected with cDNA coding for GFP, GFP-PEDF wt and the following mutants: GFP-PEDF^{K146A-K147A-R149A}; GFP-PEDF^K48A-K53A; GFP-PEDF^{Y63F-Y66F-R67Q-R69Q-K70A}. A. Transiently transfected cells were fixed, stained with DAPI and analyzed by confocal microscopy. As negative control for nuclear import, cells were transiently transfected with GFP-crm-A. B. Nuclear localization of GFP, GFP-PEDF and mutants was assessed in transiently transfected cells as a ratio of nuclear to cytoplasmic fluorescence using the Zeiss LSM 510 software. Data are means +/− SD from n = 16 fluorescent cells analyzed. * p<0.05 difference from GFP-PEDF transfected cells. Scale bar = 20 µm. C. Cells transiently transfected with GFP-PEDF and mutants were subjected to subcellular fractionation. The cytosolic and nuclear fractions were subjected to immunoblotting using a polyclonal GFP antibody. A polyclonal anti-lamin and a monoclonal anti-actin antibody were used as nuclear and cytoplasmic controls respectively. D. Cytosolic and nuclear fractions from HUVEC cells and mouse liver tissue, subjected to immunoblotting with anti-PEDF polyclonal antibody to detect nuclear localisation of endogenous PEDF.</p

Identification of putative PEDF nuclear import motifs.

<p>A. Alignment of PEDF with the known transportin-SR2 substrate cargo RNA binding protein, RBM4b (Genbank Acc. AAH04951). The RBM4b C-terminal domain (amino acids 196–364) interacts with TRN-SR2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026234#pone.0026234-Lai2" target="_blank">[20]</a>. B. Potential NLS residues highlighted in the crystal structure of PEDF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026234#pone.0026234-Simonovic1" target="_blank">[24]</a>. The novel YxxYRVRS motif is found in helix A (green), with possible linked bipartite lysine residues in yellow. The basic residues important for heparin binding are shown in red.</p

Arginine 67 and 69 are required for PEDF interaction with Transportin-SR2.

<p>GST and GST-TRN-SR2 were purified on glutathione-agarose from BL21 <i>E.coli</i>. Human recombinant PEDF and PEDF^R67Q-R69Q were purified on heparin-agarose from medium of transiently transfected HEK293T cells. GST or GST-TRN-SR2 were incubated with glutathione-agarose for 1 hr followed by addition of recombinant PEDF or PEDF^R67Q-R69Q for 4 h at 4°C. Samples were subjected to immunoblotting using a monoclonal GST, a monoclonal PEDF and a monoclonal Transportin-SR2 antibody.</p

Arginine 67 and 69 are required for PEDF nuclear accumulation in transiently and stably transfected cells.

<p>HEK293T cells were transiently transfected with cDNA coding for GFP-PEDF and GFP-PEDF^R67Q-R69Q. Transfected cells were incubated with G418 (500 µg/ml) for 14 days and stable transfected fluorescent cells were enriched by fluorescence-activated cell sorting (FACS). A. Transiently transfected cells were fixed, stained with DAPI and analysed by confocal microscopy. Scale bar = 20 µm. B, C. Stably transfected cells were lysed and subjected to immunoblotting using a polyclonal GFP and a monoclonal PEDF antibody. D, E. Stably transfected cells were fixed, stained with DAPI and analyzed by confocal microscopy. 40× images were collected and nuclear localisation of GFP, GFP-PEDF and GFP- PEDF^R67Q-R69Q was expressed as ratio of nuclear to cytoplasmic fluorescence. Data expressed as mean +/− SD from n = 200 cells analyzed. * p<0.05 difference from GFP-PEDF transfected cells. F, G. Control and stably transfected cells were incubated with vehicle (PBS) or permeabilized with digitonin (2 µg/ml) for 10 min at 4°C. Fluorescent emission was acquired using flow cytometry. Data are means +/− SD from n = 3 separate experiments. * p<0.05 difference from GFP-PEDF transfected cells. Scale bar = 20 µm.</p

Primer Sequences.

<p>Primer Sequences.</p

Transportin SR2 knock down inhibits nuclear accumulation of GFP-PEDF.

<p>HEK293T cells stably transfected with cDNA coding for GFP, GFP-PEDF and GFP-PEDF^R67Q-R69Q were transfected with a scrambled siRNA (ctrl siRNA) or with a Transportin SR2 siRNA (TRN-SR2 siRNA). After 48 hours cells were fixed, stained with DAPI and analyzed by confocal microscopy. 63× images were collected and nuclear localisation of GFP, GFP-PEDF and GFP- PEDF^R67Q-R69Q was observed. Scale bar = 20 µm.</p