Figure 1
Figure 1. CpdA inhibits p27Kip1 ubiquitination and turnover. (A) An in vitro–reconstituted system was developed that was capable of modifying labeled p27 (lane 1, 4) into polyubiquinated forms (lane 2). In the presence of 30 μM CpdA, ubiquitination of p27 was inhibited (lane 3). Image J software (http://rsb.info.nih.gov/ij/) measurement showed that CpdA treatment reduced the level of polyubiquitinated p27 to 57% compared with that seen in the vehicle-treated sample (lane 2). Addition of excess Skp2, Skp1, Cul1, and Roc1 restored p27 ubiquination both in the absence of CpdA (lane 5) and in its presence (lane 6), thereby overcoming the activity of Cpd A. (B) The structure of CpdA. (C) HeLa cells arrested in G1/S phase by double thymidine block were treated with DMSO, 30 μM CpdA, or 10 μM MG132 for 16 hours. Cycloheximide (CHX) was then added for the indicated times, and cell lysates were analyzed by Western blotting to detect p27 and Thr187–phospho-p27. β-actin served as a loading control. (D) HeLa cells were transfected with either a control shRNA (shneg), or an shRNA targeting Skp2 (shSkp2). They were then treated with either vehicle or CpdA for 24 hours, and analyzed by Western blotting for their content of the indicated proteins. (E) HeLa cells were transfected with pcDNA3.0 or pcDNA3.0-HA-Skp1, and then treated with vehicle or 20 μM CpdA for 4 hours. Whole-cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody and probed for their Skp2 content. The location of Skp2 and the heavy chain of the precipitating antibody (IgH) are indicated at the right. As a loading control, 50 μg whole-cell lysates were probed with anti-Skp2 and anti-Skp1 antibodies. (F) WT, p27−/−, and Skp2−/− MEF cells were treated with vehicle or 5 μM CpdA for 24 hours, and analyzed by Western blotting for their content of the indicated proteins. (G) WT, p27−/−, and Skp2−/− MEF cells were treated with vehicle or either 20 μM or 30 μM CpdA for 24 hours; cell viability was assessed using the WST-1 assay. The data shown represent the mean plus or minus standard deviation (SD) of triplicate cultures.

CpdA inhibits p27Kip1 ubiquitination and turnover. (A) An in vitro–reconstituted system was developed that was capable of modifying labeled p27 (lane 1, 4) into polyubiquinated forms (lane 2). In the presence of 30 μM CpdA, ubiquitination of p27 was inhibited (lane 3). Image J software (http://rsb.info.nih.gov/ij/) measurement showed that CpdA treatment reduced the level of polyubiquitinated p27 to 57% compared with that seen in the vehicle-treated sample (lane 2). Addition of excess Skp2, Skp1, Cul1, and Roc1 restored p27 ubiquination both in the absence of CpdA (lane 5) and in its presence (lane 6), thereby overcoming the activity of Cpd A. (B) The structure of CpdA. (C) HeLa cells arrested in G1/S phase by double thymidine block were treated with DMSO, 30 μM CpdA, or 10 μM MG132 for 16 hours. Cycloheximide (CHX) was then added for the indicated times, and cell lysates were analyzed by Western blotting to detect p27 and Thr187–phospho-p27. β-actin served as a loading control. (D) HeLa cells were transfected with either a control shRNA (shneg), or an shRNA targeting Skp2 (shSkp2). They were then treated with either vehicle or CpdA for 24 hours, and analyzed by Western blotting for their content of the indicated proteins. (E) HeLa cells were transfected with pcDNA3.0 or pcDNA3.0-HA-Skp1, and then treated with vehicle or 20 μM CpdA for 4 hours. Whole-cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody and probed for their Skp2 content. The location of Skp2 and the heavy chain of the precipitating antibody (IgH) are indicated at the right. As a loading control, 50 μg whole-cell lysates were probed with anti-Skp2 and anti-Skp1 antibodies. (F) WT, p27−/−, and Skp2−/− MEF cells were treated with vehicle or 5 μM CpdA for 24 hours, and analyzed by Western blotting for their content of the indicated proteins. (G) WT, p27−/−, and Skp2−/− MEF cells were treated with vehicle or either 20 μM or 30 μM CpdA for 24 hours; cell viability was assessed using the WST-1 assay. The data shown represent the mean plus or minus standard deviation (SD) of triplicate cultures.

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