Journal: Human molecular genetics

Article Title: Protein kinase C gamma, a protein causative for dominant ataxia, negatively regulates nuclear import of recessive-ataxia-related aprataxin.

doi: 10.1093/hmg/ddp298

Figure Lengend Snippet: Figure 3. Impaired nuclear localization of APTX by mutant PKCg and interaction of these proteins. (A) Coexpression of WT or the common mutant S119F-dsRed-PKCg with various GFP-fused proteins in SH-SY5Y cells. The upper panels (fluorescent images and a graph) show APTX and other neurodegen- erative disease-causing proteins, although the lower panel (a graph) shows nuclear proteins. Nuclear accumulations of GFP-APTX were significantly lower in cells expressing S119F-PKCg than in WT-PKCg transfection cells, whereas other GFP-fused proteins showed similar localization patterns in both WT and S119F-PKCg transfection cells. P , 0.05, unpaired Student’s t-test as compared with WT transfection cells. (B) Effects of mutant (S119F and M697Iex) PKCg on endogenous APTX. SH-SY5Y cells were separately transfected with WT-, S119F- and M697Iex-GFP-PKCg plasmids. Endogenous APTX was immu- nostained with anti-APTX monoclonal antibody and Alexa594-secondary antibody (red). Nuclear accumulation of APTX was reduced in cells expressing mutant PKCg, but not WT-PKCg. The bar represents the mean+SD from five independent experiments. P , 0.05, unpaired Student’s t-test as compared with mock transfection cells. (C) Interaction of WT/mutant PKCg and endogenous APTX. Immunoprecipitation (IP) with anti-GFP antibody followed by western blot (WB) with anti-APTX and anti-GFP antibodies showed interaction of endogenous APTX with WT/mutant GFP-PKCg expressed in SH-SY5Y cells (upper left panels). The converse experiment confirmed the results (upper right panels). KD PKCg proteins considerably decreased the interaction. Nonspecific interactions of endogenous APTX or GFP-PKCg with rabbit or mouse IgG were excluded by IP with nonspecific IgG (lower panels).

Article Snippet: Immunoprecipitation of endogenous APTX with the monoclonal antibody and subsequent immunoblotting with anti-GFP antibody and rabbit polyclonal anti-APTX antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) was conversely performed.

Techniques: Mutagenesis, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: Human molecular genetics

Article Title: Protein kinase C gamma, a protein causative for dominant ataxia, negatively regulates nuclear import of recessive-ataxia-related aprataxin.

doi: 10.1093/hmg/ddp298

Figure Lengend Snippet: Figure 4. Identification of a phosphorylated residue of APTX and the mechanism inhibiting its nuclear entry. (A) Coomassie blue staining showed equivalent amounts of WT-, T111A- and S118A-APTX used as substrates for in vitro kinase assay (upper panel). Km values of WT- and mutant (S119F and M697Iex) PKCg, using recombinant WT-APTX substrate, are shown above the autoradiographic images. Autoradiograms of the electrophoresed end products after com- plete phosphorylation revealed that both WT- and mutant (S119F and M697Iex) PKCg phosphorylated WT- and S118A-APTX, but not T111A-APTX (middle panels). Similarly, phosphorylation of peptides around the Thr111 without Ser118 residue was seen in WT-APTX, but not in T111A-APTX (lower panel). (B) WT-dsRed-APTX was localized to the DAPI-stained nuclei in SH-SY5Ycells without PKCg expression, but was localized to the cytoplasm in S119F-GFP-PKCg transfected cells. The decreased nuclear accumulations of APTX were restored by calphostin C (Cal), a selective PKC inhibitor. S118A-APTX, but not T111A mutant, showed nuclear import failure in S119F-PKCg transfected cells. T111E-APTX, mimicking phosphorylated APTX, reduced nuclear import without PKCg expression. KD-S119F PKCg did not affect the nuclear import. M697Iex had slightly mild effects on APTX nuclear accumulation, as shown in the graph. The bar represents the mean+SD from five independent experiments. P , 0.05, unpaired Student’s t-test as compared with the sample for WT-APTX transfection alone. (C) In vivo phosphorylation of APTX in SH-SY5Y cells. WT- and T111A-GFP-APTX plasmids were sep- arately cotransfected with WT-, S119F- or M697Iex-dsRed-PKCg plasmid, and were labeled with [32P]phosphate. Cytoplasmic (C) and nuclear (N)-fractionated and immunoprecipitation (IP)-purified proteins were subjected to autoradiography and western blotting (WB). WT-APTX was phosphorylated and present in the cytoplasm, although T111A-APTX was not phosphorylated and was present in the nucleus (upper panels). Mutant PKCg (S119F and M697Iex) phosphorylated WT-APTX considerably more than did WT-PKCg. Calphostin (Cal) inhibited phosphorylation and promoted nuclear entry of WT-APTX. (D) Impaired inter- action between phosphorylated APTX and the nuclear import adaptor importin a. Immunoprecipitation assay showed that importin a interacted with unpho- sphorylated WT-APTX and phosphorylation-resistant T111A-APTX, but had significantly reduced interactions with WT- and S118A-APTX phosphorylated by mutant (S119F and M697Iex) PKCg and a mildly reduced interaction with phosphomimetic T111E-APTX.

Article Snippet: Immunoprecipitation of endogenous APTX with the monoclonal antibody and subsequent immunoblotting with anti-GFP antibody and rabbit polyclonal anti-APTX antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) was conversely performed.

Techniques: Residue, Staining, In Vitro, Kinase Assay, Mutagenesis, Recombinant, Phospho-proteomics, Expressing, Transfection, In Vivo, Plasmid Preparation, Labeling, Immunoprecipitation, Autoradiography, Western Blot

Journal: Human molecular genetics

Article Title: Protein kinase C gamma, a protein causative for dominant ataxia, negatively regulates nuclear import of recessive-ataxia-related aprataxin.

doi: 10.1093/hmg/ddp298

Figure Lengend Snippet: Figure 5. Effects of APTX phosphorylation on DNA damage and cell death. (A) DNA damage in SH-SY5Y cells expressing mutant PKCg and effects of various APTX mutants. Cells were transfected with dsRed-WT/T111A/T111E/ S118A-APTX/LacZ gene [APTX(-)] with or without untagged mutant (S119F) PKCg cDNA, and treated with 1 mM BSO for 10 h, without cell death. An oxida- tively damaged nucleotide (8-oxoguanine: 8-oxoG) was immunostained and visu- alized with FITC (green). Cells with reduced nuclear localization of WT- and S118A-APTX with mutant PKCg showed nuclear accumulation of 8-oxoG. T111E without mutant PKCg mildly reduced nuclear APTX and increased 8-oxoG. Treatment with the kinase inhibitor calphostin C (Cal) increased nuclear APTX and reduced 8-oxoG. In contrast, treatment with the antioxidant decylubiquinone (DEC) did not affect APTX localization, but reduced 8-oxoG. M697Iex had milder effects on 8-oxoG accumulation as shown in the graph. The bar represents the mean+SD from five independent experiments. P , 0.05, unpaired Student’s t-test as compared with a sample for dsRed-WT-APTX transfection and BSO-treatment. (B) Calphostin C dose-dependently blocked cell death caused by 1 mM BSO for 24 h in SH-SY5Y cells expressing M697Iex and S119F mutant PKCg. P , 0.01, Mann–Whitney U test as compared with no calphostin C treatment. (C) BSO-induced cell death for mutant (S119F and M697Iex) PKCg expression was also blocked by coexpression of phosphorylation-resistant T111A-APTX and by treatments with antioxidants (DEC and all-trans-retinol [VA]), but not by WT or S118A-APTX or by parkin expression. Treatment with the caspase 12 inhibitor z-ATAD-fmk had also no effect on cell viability. P , 0.01, Mann–Whitney U test as compared with expression of mutant (S119F or M697Iex) PKCg alone.

Article Snippet: Immunoprecipitation of endogenous APTX with the monoclonal antibody and subsequent immunoblotting with anti-GFP antibody and rabbit polyclonal anti-APTX antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) was conversely performed.

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Transfection, MANN-WHITNEY

Journal: Human molecular genetics

Article Title: Protein kinase C gamma, a protein causative for dominant ataxia, negatively regulates nuclear import of recessive-ataxia-related aprataxin.

doi: 10.1093/hmg/ddp298

Figure Lengend Snippet: Figure 6. Distribution of APTX in neurons. (A) Pathological specimens of the control human cerebellum. Conventional horseradish peroxidase-based immu- nostaining (without nuclear counterstaining) showed that Purkinje cells had high levels of PKCg and APTX predominantly in the cytoplasm with small amounts of both proteins in nuclei (first and second columns). Coimmunostaining followed by confocal microscopic imaging reproduced the results (remaining columns). Scale bar represents 50 mm. (B) Confocal microscopic analyses of PC12 cells showed that immunostained endogenous APTX was distributed in the nucleus in undifferentiated cells, but in both the cytoplasm and nucleus in differentiated cells. BSO treatment (1 mM for 12 h without cell death) as well as dsRed-M697Iex expression by transfection decreased nuclear APTX in differentiated PC12 cells. Calphostin C (Cal) increased nuclear APTX. Scale bar rep- resents 10 mm. P , 0.05, unpaired Student’s t-test as compared with the sample for undifferentiated PC12 cells without BSO treatment. #P , 0.05, unpaired Student’s t-test between samples for differentiated PC12 cells as compared with the sample without BSO, Cal or M697Iex. (C) Immunoblot showed that differ- entiation increased PKCg and APTX levels in PC12 cells, with a larger effect on PKCg. P , 0.05, unpaired Student’s t-test as compared with the sample for undifferentiated PC12 cells.

Article Snippet: Immunoprecipitation of endogenous APTX with the monoclonal antibody and subsequent immunoblotting with anti-GFP antibody and rabbit polyclonal anti-APTX antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA) was conversely performed.

Techniques: Control, Imaging, Expressing, Transfection, Western Blot