The amino-terminus of ADAR2 harbours a Pin1-binding site. (A) The anti-MPM-2 antibody recognizes potential Pin1 sites in ADAR2 purified after overexpression in P. pastoris. Immunoblot analysis with anti-MPM-2 antibody of anti-FLAG immunoprecipitates from lysates of HEK293T cells transfected with FLAG-tagged hADAR2, ADAR2^S26A/S31A, ADAR2^{S26A/S31A/T32A}, ADAR2^S26A, ADAR2^T32A or pcD3. The minor band in the lane with pcD3 is contamination from the neighbouring lane. ADAR input visualized with anti-FLAG antibody, lower panel. (B) Purified ADAR2 binds in vitro to Pin1 immobilized on beads. (Upper left panel) Immunoblot analysis with anti-FLAG antibody of the binding of FLAG-tagged ADAR2, ADAR2^S26A, ADAR2^{S26A/S31A/T32A}, ADAR2^Δ4–72 bound to GST–Pin1 or GST on glutathione beads. (Lower panel) GST input visualized with anti-GST antibody. (Right panel) Purified ADAR proteins stained with Coomassie. (C) Binding of purified ADAR2 to Pin1 depends on phosphorylation of Pin1 sites on ADAR2. λ phosphatase treatment of lysate from HEK293T cells transfected with ADAR2 for 0 (−), 2 h (+), 3 h (++) prior to incubation with GST–Pin1. Immunoblot analysis of ADAR2 with anti-FLAG antibody. Middle and lower panels are input loading controls. (D) Pin1 binds to ADAR2 in HEK293T cells. Coimmunoprecipitation of ADAR2 and Pin1 performed with anti-FLAG antibody on HEK293T cell lysate cotransfected with HA–Pin1 and either FLAG-tagged ADAR2, ADAR2^Δ4–72, ADAR2^RRM1–2, ADAR2^{S26A/S31A/T32A} or pcD3. HA–Pin1 was detected with anti-HA antibody. Asterisks represent IgG light chain. (Lower panel) Immunoblot of input proteins with anti-FLAG antibody. (E) Endogenous Pin1 detected with anti-Pin1 antibody after immunoprecipitation with anti-FLAG antibody from cell lysates of HEK293T cells transfected with FLAG-tagged ADAR2, ADAR2^{S26A/S31A/T32A}, ADAR2^Δ4–72, ADAR2^RRM1–2 or pcD3. (Lower panel) Immunoblot of input proteins detected with anti-FLAG antibody. Asterisks represent IgG light chain.

Pin1 is required for nuclear localization of ADAR2. (A) ADAR2 is mislocalized from the nucleus to the cytoplasm in Pin1^−/− MEF cells. GFP–ADAR2 immunofluorescence in Pin1^+/+ and Pin1^−/− MEF cells cotransfected with GluR2 B13 minigene and GFP–ADAR2. DAPI staining of nuclei (i, iv), GFP fluorescence of cell (ii, v) and merged (iii, vi). (B) Nuclear localization of ADAR2 is restored in Pin1^−/− MEF cells by transfection of HA–Pin1. GFP–ADAR2 (green) direct and HA–Pin1 (red) indirect immunofluorescence detection in Pin1^−/− MEF cells cotransfected with GluR2 B13 minigene and (ii) GFP–ADAR2 (green) and (iii) HA–Pin (red). (i) DAPI staining of nuclei. (iv) Merge of all three images. (C) Nuclear localization of ADAR2 depends on catalytic activity of Pin1. GFP–ADAR2 (green) and HA–Pin1^S67E (red) in Pin1^−/− MEF cells cotransfected with GluR2 B13 minigene and (ii) GFP–ADAR2 (green) and (iii) HA–Pin1^S67E (red). (i) DAPI staining of nuclei. (iv) Merge of all three images. All photographs were taken at the same exposure. Scale bar, 10 μm. (D) Nucleo-cytoplasmic fractionation. Immunoblot analysis with anti-FLAG antibody of nuclear and cytoplasmic fractions of Pin1^−/− MEF cells transfected with FLAG–ADAR2 (lanes 1 and 2). Pin1 was cotransfected with FLAG–ADAR2 in Pin1^−/− MEF cells (lanes 3 and 4). HA–Pin1^S67E was cotransfected in Pin1^−/− MEF cells (lanes 5 and 6). (Lower panel) Immunoblot of fractionated Pin1^−/− MEF cells with tubulin as a marker for cytoplasmic fraction and HP1α for nuclear fraction.

Pin1 contributes to stability of ADAR2 protein. (A) Knockdown of Pin1 in HeLa cells destabilizes ADAR2 in a cycloheximide time course. HeLa cells were transfected with either pSuper LacZ or pSuper Pin1. Cycloheximide (50 μg/μl) was added to both and a time course from 0 to 8 h was performed. Cell lysates were analysed by immunoblot and the antibodies used were anti-ADAR2 (top panel), anti-tubulin as a loading control (middle panel) and Pin1 (bottom panel). (B) Quantification of (A). (C) Pin1 knockdown destabilization of FLAG–ADAR2 in SH-SY5Y neuroblastoma cells. SH-SY5Y cells were cotransfected with FLAG-tagged ADAR2 and a control siRNA or Pin1-specific siRNA. Cycloheximide (50 μg/μl) was added to both and a time course from 0 to 8 h was performed. Cell lysates were analysed by immunoblot and the antibodies used were anti-FLAG (top panel), anti-Pin1 (middle panel) and GFP as loading control (bottom panel). (D) ADAR2 mutant in the Pin1-binding site is less stable. SH-SY5Y neuroblastoma cells were transfected with FLAG-tagged ADAR2^{S26A/S31A/T32A} and cycloheximide (50 μg/μl) was added and a time course was performed from 0 to 8 h. Cell lysates were analysed by immunoblot and the antibodies used were anti-FLAG (top panel), anti-Pin1 (middle panel) and GFP as loading control (bottom panel).

WWP2 is required for ADAR2 ubiquitination and subsequent degradation in the cytoplasm. (A) In vivo ubiquitination assays. FLAG–ADAR2–His, FLAG–WWP2–His and V5-UBQ were transfected in HEK293T cells followed by purification of ubiquitination complexes from lysates with Ni²⁺-NTA. In lane 1, cotransfection was with FLAG–ADAR2–His, FLAG–WWP2–His. Lane 2, cotransfection was with FLAG–ADAR2–His, FLAG–WWP2–His and V5-UBQ. Lane 3, cotransfection of FLAG–ADAR2 NH₂^–PPxY–His, FLAG–WWP2–His and V5-UBQ. Lane 4, cotransfection of FLAG–ADAR2 COOH^–PPxY–His, FLAG–WWP2–His and V5-UBQ. Lane 5, cotransfection of FLAG–ADAR2^–PPxY–His (double mutant), FLAG–WWP2–His and V5-UBQ. Lane 6 is the same as lane 5 without the addition of V5-UBQ and is the negative control. (Middle panel) Immunoblot of input proteins detected with anti-FLAG antibody. (Lower panel) Immunoblot of V5-UBQ present in the purified complex detected with anti-V5 antibody. (B) Immunoblot at 24 h following transfection of FLAG–ADAR2 and FLAG–ADAR2^–PPxY with 20 μM MG132 to inhibit protein degradation. The proteasomal inhibitor MG132 was added to both and a time course from 0 to 4.5 h was performed in HeLa cells. Cell lysates were normalized to GFP levels (lower panel). (C) Immunofluorescence of Pin1^+/+and Pin1^−/− MEF cells cotransfected with GluR2 and FLAG–ADAR2^–PPxY (double mutant). (i) DAPI staining of nuclei. (ii) Anti-FLAG–ADAR2^–PPxY (red). (iii) Merge of DAP1 and FLAG. Scale bar, 10 μm. All photographs were taken at the same exposure. (D) Nuclear and cytoplasmic fractionation of FLAG–ADAR2^–PPxY in Pin1^+/+and Pin1^−/− MEF cells. (Lower panels) Immunoblot of fractionated MEF cells with tubulin as a cytoplasmic marker and HP1α as a nuclear marker. (E) Quantification of (D).

Pin1 is required for efficient editing at the GluR2 Q/R site. (A) DNA sequence chromatograph of the RT–PCR product of the region encompassing the Q/R site (arrow) encoded by the GluR2 B13 minigene transiently cotransfected with ADAR2 (2 μg) in HeLa cells, editing is 100% (left chromatograph). Editing of the Q/R site drops to 53% when an siRNA specific for Pin1 is cotransfected together with plasmids encoding both ADAR2 and the GluR2 B13 minigene (middle chromatograph). Editing is 100% at the GluR2 Q/R site when a control siRNA is cotransfected (right chromatograph). Immunoblot analysis of cell lysate from HeLa cells with either anti-Pin1 or anti-tubulin antibodies (right panel). (B) (Left panel) Sequencing chromatogram of editing by endogenous ADAR2 at the Q/R site of RT–PCR product pools from the GluR2 B13 minigene transcript that has been transiently transfected into HeLa cells. Arrows indicate Q/R editing site in all panels. Immunoblot analysis with anti-Pin1 antibody of HeLa cell extracts that have been cotransfected with GFP in the presence of either Pin1-specific siRNA, no siRNA or HA–Pin1 construct (0.5 μg). Proteins are detected with anti-Pin1 antibody and anti-GFP antibody as a loading control (right panel). (C) Chromatograph of editing of endogenous GluR2 transcript at the Q/R site in neuroblastoma SH-SY5Y cells transfected with ADAR2 (2 μg). Editing is 100% at the Q/R site (left chromatograph). A decrease in editing is observed when an siRNA specific for Pin1 was cotransfected (middle chromatograph). A control siRNA does not affect editing when transfected (right chromatograph). Arrows indicate the Q/R site. Cell lysates of SH-SY5Y cells were analysed by immunoblot with either anti-Pin1 or anti-tubulin (right panel). (D) Chromatograph of editing at the Q/R site of GluR2 B13 minigene transcript in Pin1^−/− MEF cells transfected with ADAR2 (2 μg). Editing increased to 100% when Pin1^−/− MEF cells were cotransfected with either 0.5 or 1 μg of a construct expressing Pin1. An arrow marks the Q/R editing site in all the chromatographs.

Proline 33 of ADAR2 is required for the Pin1 effect on ADAR2 nuclear localization. (A) Normal localization of ADAR2 and Pin1. Immunofluorescence of HeLa cells cotransfected with GluR2 B13 minigene, FLAG–ADAR2 and HA–Pin1 stained with (i) DAPI, (ii) anti-HA–Pin1 (green), (iii) anti-FLAG–ADAR2 (red), (iv) Merge of DAP1 and FLAG and (v) merge of all three images. (B) Nuclear localization of ADAR2 depends on Proline 33. Immunofluorescence of HeLa cells cotransfected with GluR2 B13 minigene, FLAG–ADAR2^P33A and HA–Pin1 stained with (i) DAPI, (ii) anti-HA–Pin1 (green), (iii) anti-FLAG–ADAR2^P33A (red), (iv) Merge of DAP1 and FLAG and (v) merge of all three images. All photographs were taken at the same exposure. Scale bar, 10 μm. (C) Nucleo-cytoplasmic fractionation of wild-type and ADAR^P27A and ADAR2^P33A mutants. Immunoblot analysis with anti-FLAG antibody of nuclear and cytoplasmic fractions of HeLa cells transfected with FLAG–ADAR2 (lanes 1 and 2), ADAR^P27A (lanes 3 and 4), ADAR2^P33A (lanes 5 and 6). (Lower panels) Immunoblot of fractionated HeLa cells with tubulin as a cytoplasmic marker and HP1α as a nuclear marker.

WWP2 interacts with ADAR2. (A) Coimmunoprecipitation of ADAR2 and WWP2 performed with anti-FLAG antibody in HEK293T cell lysate cotransfected with FLAG–ADAR2 and c-myc–WWP2 or pcD3 empty vector. FLAG–ADAR2 was detected with FLAG antibody. WWP2 input visualized with anti-c-myc antibody (bottom panel). (B) Endogenous WWP2 detected with anti-WWP2 antibody after immunoprecipitation with anti-FLAG antibody from lysates of HEK293T cells transfected with FLAG-tagged ADAR2, ADAR2^2–305, ADAR2^298–701, ADAR2^Δ4–72 or pcD3. Immunoblot of input proteins detected with anti-FLAG antibody (bottom panel). (C) Immunoprecipitation of FLAG–ADAR2 and mutants with mutations in the amino, carboxyl binding site for WWP2 or a combination of both was performed with anti-FLAG antibody in HEK293 cells. Endogenous WWP2 detected with anti-WWP2 antibody. (Lower panel) Immunoblot of input proteins detected with anti-FLAG antibody. (D) Immunoblot analysis with anti-FLAG antibody of lysates from HeLa cells cotransfected with FLAG–ADAR2 and increasing amount of FLAG–WWP2 (0.5, 1, 2.5 μg) (upper panel) and this experiment was repeated with the mutant ADAR2^PPxY that is unable to bind to WWP2 (lower panel). The efficiency of transfection was normalized to GFP expression. GFP input visualized with anti-GFP antibody is shown below both panels.

Schematic representation of the regulation of ADAR2 by Pin1 and WWP2. ADAR2 can exist as a monomer in the nucleus and has sequences at its amino-terminus that inhibit enzymatic activity (Macbeth et al, 2004). However, ADAR2 can be phosphorylated by an unknown kinase either when it is free or bound to dsRNA. ADAR2 is a substrate for Pin1 once it is bound to dsRNA and the active form of ADAR2 is a dimer (Gallo et al, 2003; Poulsen et al, 2006; Valente and Nishikura, 2007). The mechanism of dimer formation is still unclear. After RNA editing has occurred, we do not know the fate of ADAR2. However, in the absence of Pin1, ADAR2 mislocalizes to the cytoplasm where it is poly-ubiquitinated by WWP2 and is degraded by the proteasome.