Ubiquitin- and ATP-Independent Proteolytic Turnover of p21 by the REGγ-Proteasome Pathway

Abstract

We previously demonstrated that the proteasome activator REGgamma directs degradation of the steroid receptor coactivator SRC-3 by the 20S proteasome in an ATP- and ubiquitin-independent manner. Our efforts to identify additional endogenous direct targets of the REGgamma proteasome revealed that p21(Waf/Cip1), a central cyclin-dependent kinase inhibitor, is another endogenous target. Gain-of-function analysis, RNAi knockdown, REGgamma-deficient MEF analysis, and pulse-chase experiments substantiate that REGgamma promotes degradation of unbound p21. Cell-free proteasome proteolysis assays using purified REGgamma, p21, and the 20S proteasome confirm that REGgamma directly mediates degradation of free p21 in an ATP- and ubiquitin-independent manner. Depletion of REGgamma in a thyroid carcinoma cell line results in cell-cycle and proliferative alterations. Our study reveals that, in addition to degrading the SRC-3 growth coactivator, REGgamma also has a role in the regulation of the cell cycle through its ability to influence the level of a cell-cycle regulator(s).

Regulation of c-Myc protein stability by proteasome activator REGγ

“…We further examined the regulation of c-Myc by REGγ using REGγ − / − (REGγ knockout) murine embryonic fibroblasts (MEFs). As expected, REGγ deficiency significantly increased the protein levels of c-Myc as well as the reported REGγ substrates p53 and p21, 10,30 but had no measurable effect on the protein levels of AKT and LSD1 (Figure 2a). This result was confirmed using an immunofluorescence staining assay (Figure 2b).…”

“…6 REGγ is emerging as an alternative pathway to target intact proteins for degradation independent of the 26S proteasome. 7,30 Thus, we examined whether REGγ has any role in c-Myc protein turnover. Our data showed that ectopic expression of REGγ resulted in a marked reduction in c-Myc protein in a dosedependent manner (Figure 1b).…”

“…7 Following that finding, more proteins, such as p21, ARF, p14, MAFA, and AID, were found to be degraded by REGγ. 8,30,43,44 Interestingly, REGγ can degrade oncoproteins including SRC-3 and PTTG1, as well as tumor suppressors including p21, ARF and p53. [7][8][9][10] Thus, it is expected that REGγ has a context-dependent role in tumorigenesis.…”

“…The expression plasmids of WT and N151Y REGγ were described previously. 30 RhoA promoter reporter was kindly provided by Dr. Hui-Kuan Lin. 37 E2F1 promoter reporter was kindly provided by Dr. Mu-Shui Dai.…”

“…Immortalized MEFs were described in a previous report. 30 All cell lines were cultured in DMEM supplemented with 10% FBS (HyClone, Logan, UT, USA). Transfections were performed using the calcium phosphate-DNA co-precipitation method for HEK293T cells, and SunbioTrans-EZ for HeLa cells.…”