Journal: Cell Death and Differentiation
Article Title: Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death
doi: 10.1038/cdd.2015.103
Figure Lengend Snippet: Cdk5 phosphorylates CHIP at Ser20. (a) Recombinant GST-CHIP protein (3 μg) was incubated with the purified active Cdk5/p25 kinase complex (0.5 μg) in the presence of [γ-32P]ATP plus indicated doses of roscovitine, a selective Cdk5 inhibitor. The reaction products were resolved by SDS-PAGE and subjected to autoradiography. Coomassie brilliant blue (CBB) staining for GST-CHIP protein was provided at the bottom and used as a loading control. (b) HEK293 cells were transfected with HA-CHIP in combination with either Flag-Cdk5 WT/p25-Myc or Flag-Cdk5 KD/p25-Myc. Cell lysates were resolved by SDS-PAGE followed by immunoblot (IB) analysis using the indicated antibodies. Positions of the phosphorylated and non-phosphorylated HA-CHIP were indicated by the white dotted lines. Actin antibody was used as a loading control. (c) Following transfection of HEK293 cells with HA-CHIP and Flag-Cdk5/p25-Myc, cell lysates were incubated with calf intestinal alkaline phosphatase (CIP). The reaction products were subjected to IB analysis using the indicated antibodies. (d) Schematic representation of the putative phosphorylation sites on CHIP obtained from bioinformatic analyses through the computational databases depicted in Supplementary Table 1. Three putative serine (S) phosphorylation sites (red) and two putative threonine (T) phosphorylation sites (green) are indicated. TPR, tetratricopeptide repeat domain; +/−, highly charged domain; U-box, E3 ligase domain. (e) Recombinant GST-CHIP WT (3 μg) or other phospho-null mutant proteins (S20A, S24A, T247A, S274A and T277A; 3 μg each) was incubated with the purified, active Cdk5/p25 kinase complex (0.5 μg) in the presence of [γ-32P]ATP. The reaction products were resolved by SDS-PAGE and subjected to autoradiography. (f) Antibodies specifically recognizing the phosphorylated Ser20 site (pS20 CHIP) were raised from the peptide sequences, TGGG(pS)PDKSP surrounding the Ser20 residue of mouse CHIP. The specificity of the antibody was confirmed by the absence of the corresponding band in immunoprecipitation (IP) analysis using the cell lysates from HEK293 cells transiently expressing HA-CHIP S20A in combination with Flag-Cdk5 WT/p25-Myc when compared with that of wild type
Article Snippet: Preparation of anti-pS20 CHIP antibody The production of anti-Ser20 CHIP phospho-specific rabbit polyclonal antibody was designed in our laboratory and generated by Abmart (Shanghai, China) using peptide C-TGGGG(pS)PDKSP surrounding the serine 20 residue on mouse CHIP.
Techniques: Recombinant, Incubation, Purification, SDS Page, Autoradiography, Staining, Control, Transfection, Western Blot, Phospho-proteomics, Mutagenesis, Residue, Immunoprecipitation, Expressing
![Cdk5 phosphorylates <t>CHIP</t> at Ser20. (a) Recombinant GST-CHIP protein (3 μg) was incubated with the purified active Cdk5/p25 kinase complex (0.5 μg) in the presence of [γ-32P]ATP plus indicated doses of roscovitine, a selective Cdk5 inhibitor. The reaction products were resolved by SDS-PAGE and subjected to autoradiography. Coomassie brilliant blue (CBB) staining for GST-CHIP protein was provided at the bottom and used as a loading control. (b) HEK293 cells were transfected with HA-CHIP in combination with either Flag-Cdk5 WT/p25-Myc or Flag-Cdk5 KD/p25-Myc. Cell lysates were resolved by SDS-PAGE followed by immunoblot (IB) analysis using the indicated antibodies. Positions of the phosphorylated and non-phosphorylated HA-CHIP were indicated by the white dotted lines. Actin antibody was used as a loading control. (c) Following transfection of HEK293 cells with HA-CHIP and Flag-Cdk5/p25-Myc, cell lysates were incubated with calf intestinal alkaline phosphatase (CIP). The reaction products were subjected to IB analysis using the indicated antibodies. (d) Schematic representation of the putative phosphorylation sites on CHIP obtained from bioinformatic analyses through the computational databases depicted in Supplementary Table 1. Three putative serine (S) phosphorylation sites (red) and two putative threonine (T) phosphorylation sites (green) are indicated. TPR, tetratricopeptide repeat domain; +/−, highly charged domain; U-box, E3 ligase domain. (e) Recombinant GST-CHIP WT (3 μg) or other phospho-null mutant proteins (S20A, S24A, T247A, S274A and T277A; 3 μg each) was incubated with the purified, active Cdk5/p25 kinase complex (0.5 μg) in the presence of [γ-32P]ATP. The reaction products were resolved by SDS-PAGE and subjected to autoradiography. (f) Antibodies specifically recognizing the phosphorylated Ser20 site <t>(pS20</t> CHIP) were raised from the peptide sequences, TGGG(pS)PDKSP surrounding the Ser20 residue of mouse CHIP. The specificity of the antibody was confirmed by the absence of the corresponding band in immunoprecipitation (IP) analysis using the cell lysates from HEK293 cells transiently expressing HA-CHIP S20A in combination with Flag-Cdk5 WT/p25-Myc when compared with that of wild type](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6296/pmc04716296/pmc04716296__cdd2015103f1.jpg)
![Hydrogen peroxide leads to tAIF-induced neuronal cell death via Cdk5-mediated phosphorylation of CHIP at Ser20. (a) Following treatment of primary cultures of cortical neurons with 200 μM hydrogen peroxide (H2O2) for the indicated time periods, cell lysates were harvested and subjected to IB analysis using the indicated antibodies. For in vitro kinase assay, immunoprecipitates with anti-Cdk5 antibody were reacted with 1 μg Histone H1 in the presence of [γ-32P]ATP at 30 °C for 30 min. The reaction products were resolved by SDS-PAGE and subjected to autoradiography. Coomassie brilliant blue (CBB)-stained gel represents the level of Histone H1 in each lane. (b and c) After normalization against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), relative levels of (b) pS20 CHIP and (c) tAIF were expressed as a fold intensity over the levels found in cells treated with H2O2 for 120 min (value=1). Bar represents the means±S.D. of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; NS, not significant. (d) Cell lysates harvested from hydrogen peroxide-treated cortical neurons (200 μM for 60 min) in the presence or the absence of 10 μM roscovitine were subjected to IB analysis using the indicated antibodies. In vitro kinase assay was performed as described in (a). (e) After normalization against GAPDH, relative levels of pS20 CHIP and tAIF were expressed as a fold intensity over the levels found in cells treated with H2O2 alone (value=1). Bar represents the means±S.D. of three independent experiments. **P<0.01; ***P<0.001. (f) Cortical neurons cultivated for 60 min in the indicated conditions (200 μM H2O2, 10 μM roscovitine) were subjected to the large-scale DNA fragmentation assay by using a pulsed-field gel electrophoresis. (g) Cortical neurons were exposed to 200 μM H2O2 for the indicated time periods in the presence or the absence of 10 μM roscovitine. The rate of cell viability was determined by MTT reduction assay. Values are expressed as a percent survival over the untreated control cultures (100%). Data represent the means±S.E.M. from three independent experiments done in triplicate. **P<0.01; ***P<0.001](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6296/pmc04716296/pmc04716296__cdd2015103f5.jpg)
