Volume 26, Issue 6, Pages 831-842 (June 2007) Ubiquitin- and ATP-Independent Proteolytic Turnover of p21 by the REGγ-Proteasome Pathway  Xiaotao Li, Larbi Amazit, Weiwen Long, David M. Lonard, John J. Monaco, Bert W. O'Malley  Molecular Cell  Volume 26, Issue 6, Pages 831-842 (June 2007) DOI: 10.1016/j.molcel.2007.05.028 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Overexpression of REGγ Reduced p21 Level (A) The 293-REGγ or 293-mut-REGγ cells were treated with vehicle (DMSO) or doxycycline for 24 hr. Total cell lysates were prepared for western blot analysis of REGγ and p21 levels. β-actin was probed to assess protein loading. Relative protein levels were quantitated by NIH ImageJ software and listed below each panel. (B) The 293-REGγ cells were transfected with 1 μg of pCR3.1-p21. About 12 hr posttransfection, cells were induced with 1 μg/ml of doxycycline or with vehicle for 24 hr. Pulse-chase experiments were performed as described (Li et al., 2006). Total cell lysates were extracted for immunoprecipitation, SDS-PAGE, and autoradiography. (C) Quantitated results of (B) were plotted against the indicated time course. Error bars refer to standard deviation of the average quantitated results. (D) Total RNA extracted from 293-REGγ cells treated with vehicle or doxycycline for 24 hr was prepared for real-time RT-PCR analysis. Results from three repeats were averaged and plotted as relative p21 mRNA levels. A no-RT control was included to validate this experimental procedure. Error bars refer to standard deviation of the average quantitated results. Molecular Cell 2007 26, 831-842DOI: (10.1016/j.molcel.2007.05.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Depletion of REGγ Enhanced the p21 Level in Multiple Cell Lines (A) TPC, HeLa, or LNCaP cells were treated with 20 nM siREGγ or a negative control (siNeg). Endogenous REGγ, p21, p27, or β-actin was detected by western blotting. (B) High-throughput analysis of nuclear p21 levels following RNAi in TPC cells. Automated quantitation of the average nuclear intensity of p21 was performed as described in the Experimental Procedures. The difference of nuclear accumulation of p21 between the siRNA control and siRNA-REGγ is statistically significant (p < 0.01). Error bars refer to standard deviation of the average quantitated results. (C) A representative immunofluorescence of cells treated with siREGγ. Cells successfully ablated with endogenous REGγ (pointed with white arrows) resulted in a significant increase of p21 (cells with green color). Untransfected cells displayed a high level of REGγ staining (cells with red color) and undetected p21 (pointed with red arrows). (D) Primary MEF cells were generated as described in the Experimental Procedures. Relative protein levels were quantitated and listed. Primary MEFs from the third passage were used for western blot analysis. Molecular Cell 2007 26, 831-842DOI: (10.1016/j.molcel.2007.05.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 REGγ Mediates Direct Degradation of p21 (A) REGγ and p21 interaction in vitro and in vivo. In the upper panel, GST-p21 and 35S-labeled, in vitro-translated REGγ were incubated for 4 hr at 4°C. The amount of REGγ proteins pulled down by GST-p21 was detected by SDS-PAGE and autoradiography. In the lower panel, cell lysates from HeLa cells were immunoprecipitated with anti-p21 or control IgG and detected by western blotting as described in the Experimental Procedures. (B) In the upper panel, purified REGγ, 20S proteasome and in vitro-translated p21 were incubated as indicated for 40 min at 30°C. The reaction in lane 5 contained 200 nM ATPγS, and the reaction in lane 6 was in the presence of 50 nM of epoxomicin (Epox). A fraction of the reaction was analyzed by western blotting with anti-p21 or anti-β-actin. In the lower panel, purified in vitro-translated p21 was used as a substrate for the proteolytic analysis as in the upper panel. Note that a nonspecific band (indicated by an asterisk), possibly a byproduct of GST-p21, was not degraded by the REGγ proteasome. Molecular Cell 2007 26, 831-842DOI: (10.1016/j.molcel.2007.05.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Cdk/Cyclin Complexes Can Delay REGγ-Dependent Degradation of p21 (A) In vitro proteolytic assays were performed as described in Figure 3B, except that the Cdk2/cyclin E complex or BSA was added along with p21 at the molar ratio of ∼2:1. Reactions were stopped at indicated time points by adding 5× SDS sample buffer. (B) 293-REGγ cells seated in 12-well plates and transfected with 250 ng of pCR3.1-p21 plus 1 μg of SRα296-Cdk2 and 2 μg of SRα296-cyclin A or an equal amount of control vector SRα296. Twelve hours posttransfection, cells were treated with or without 1 μg/ml doxycycline for 24 hr. Cells were then treated with 100 μg/ml cycloheximide for different periods of time as indicated. Numbers below the p21 blot indicate the relative p21 protein level of each sample relative to that at “0” time point in each individual group. Since the Cdk2 blot has indicated equal loading of individual samples, the results of the β-actin blot are not shown. Molecular Cell 2007 26, 831-842DOI: (10.1016/j.molcel.2007.05.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 REGγ-Dependent Degradation of p21 Is Not Dependent on a Functional Ubiquitin-Activating Enzyme The ubiquitin-activating enzyme temperature-sensitive cells, ts85, were transfected with control or siREGγ at 30°C. Twenty-four hours posttransfection, cells were shifted to 37°C for 20 hr followed by treatment with 100 μg/ml cycloheximide for different periods of time as indicated. An equal amount of total protein was loaded for western blotting analysis, and the relative protein levels were quantitated as described in Figure 1. Molecular Cell 2007 26, 831-842DOI: (10.1016/j.molcel.2007.05.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Effect of REGγ on Cell Cycle (A–D) A representative result of propidium iodide staining/FACScan analysis of TPC cells treated with control siRNA (A), siREGγ (B), sip21 (C), or a combination of siREGγ and sip21 (D) following RNA interference for 3 days was displayed. The averaged cell percentage and standard deviation (in parenthesis) in each phase were summarized from three individual experiments. Statistically significant differences (p < 0.05) in G2/M content (indicated by an asterisk), but not G0/G1 content, were observed between (A) and (B). (E) The BrdU incorporation of the TPC cells treated with different siRNA from three individual experiments were plotted for comparison. The data are represented as mean ± standard deviation. The statistical analysis of the results between siNeg and siREGγ, or between siNeg and siREGγ/sip21 (indicated by ∗), were all significant by paired t test (p < 0.05). (F) Western blotting demonstrates the efficient knockdown of target proteins. Molecular Cell 2007 26, 831-842DOI: (10.1016/j.molcel.2007.05.028) Copyright © 2007 Elsevier Inc. Terms and Conditions