Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome

Abstract

The cell-cycle regulator p21(Cip1) is degraded by proteasomes independently of ubiquitination. We now show that degradation of p21 in vivo does not require the 19S proteasome lid, which contains the ubiquitin-binding subunit. Instead, the major proteasomal pathway for p21 degradation involves an alternative proteasome lid, the REGgamma complex. REGgamma binds to p21 in vivo, and deletion of p21's REGgamma-binding site greatly extends its half-life. Knockdown of REGgamma by RNA interference stabilizes p21, p21 has a significantly extended half-life in REGgamma(-/-) murine embryonic fibroblasts, and the p21 abundance is elevated in REGgamma(-/-) mice. The role of REGgamma in cell-cycle regulation may extend beyond p21 regulation, because p16(INK4A) and p19(Arf) also bind to REGgamma and are stabilized in REGgamma-deficient cells.

Figure 1. p21 is degraded by the 20S proteasome

A. Equal amount of in vitro translated and ³⁵S labeled p21 were incubated with purified 20S proteasomes either alone (upper panel) or in the presence of purified cyclin E/cdk2 complexes (lower panel) at 37°C for indicated times. The reaction products were analyzed by SDS-PAGE and detected by autoradiography. B. Equal amount of in vitro translated and ³⁵S labeled p27 were incubated at 37°C for indicated times in the presence or absence of purified 20S proteasomes. C. Equal amount of in vitro translated and ³⁵S labeled p21 were incubated at 37°C for indicated times in the presence or absence of purified 26S proteasomes. Ub-Mbp-Sic1 and MeUb-Mbp-Sic1 were used as control for activity of 26S proteasomes. D. U2OS cells were transfected with siRNA against N2 subunit of the 19S proteasome. Cells were treated with cycloheximide for indicated times. GRB2 was used as a loading control for all experiments.

Figure 2. REGγ is involved in p21 degradation

A. Effects of different siRNAs against REGγ on p21 level. U2OS cells were transfected with two individual siRNAs (#1 and #2) or a pool of four siRNAs against REGγ. p21 steady state levels were determined by Western blotting. B. Effect of RNA interference of REGγ on p21 half life. U2OS cells were transfected with the siRNA pool against REGγ. Cells were treated with cycloheximide for indicated times. p21 half-lives in control U2OS cells were analyzed as well. C. Interaction between transfected p21 and endogenous REGγ. 293 cells were transfected with p21 and treated with MG-132 for 4 hr. Cell lysates were immunoprecipitated with anti-p21 or anti-REGγ antibodies, and the associated protein was detected by Western blotting. D. Interaction between endogenous p21 and REGγ. U2OS cells were treated with MG-132 for 6 hr and cell lysates were immunoprecipitated with anti-REGγ antibodies or irrelevant immune serum followed by Western blotting with anti-p21 antibodies.

Figure 3. Delineation of a REGγ interaction domain in p21

A. Interaction between endogenous REGγ and deletion mutants of p21. 293 cells were transfected with either wild type p21 or a deletion mutant as indicated. Cell lysates were immunoprecipitated with anti-REGγ antibodies followed by Western blotting with anti-p21 antibodies. B. Schematic diagram of p21 and its binding region to REGγ. (−) indicates no binding and (+) indicates binding with REGγ. C. p21 Δ156–161 binds to endogenous PCNA. 293 cells were transfected with p21, p21 Δ156–161 or p21 with mutated PCNA binding domain (p21-PCNA) and treated with MG-132 for 4 hr. Cell lysates were immunoprecipitated with anti-p21 or anti-PCNA antibodies, and the associated protein was detected by Western blotting. D. 293 cells were transfected with either p21 or p21Δ156–161. p21 half-lives were analyzed as in A.

Figure 4. p21 is stabilized in REGγ −/− MEFs

A. REGγ +/− and REGγ −/− MEFs were infected with retrovirus expressing REGγ. Steady-state levels of p21 were determined by Western blotting. B. p21 half-lives were analyzed as in Figure 2. C. REGγ −/− MEFs were infected with retrovirus expressing HA-p21K6R. p21 half-lives were analyzed as in Figure 2. D. p21 immunohistochemistry of brain tissues from wild type and REGγ −/− mice.

Figure 5. p16 degradation is ubiquitin independent

A. Equal amount of in vitro translated and ³⁵S labeled p16 were incubated at 37°C for indicated times in the presence or absence of purified 20S or 26S proteasomes. The reaction products were analyzed by SDS-PAGE and detected by autoradiography. B. p16 interacts with REGγ. 293 cells were transfected with p16. The interaction with REGγ was identified by immunoprecipitation with anti-REGγ antibodies followed by Western blotting with anti-p16 antibodies. C. Hela cells were transfected with siRNA against REGγ. p16 half-lives were analyzed as in Figure 2.

Figure 6. REGγ promotes p19^Arf turnover

A. p19^Arf interacts with REGγ. 293 cells were transfected with p19^Arf and interaction with endogenous REGγ was detected by immnoprecipitation with anti-REGγ antibodies or irrelevant immune serum followed by Western blotting with anti-p19^Arf antibodies. B. Steady-state levels of p19^Arf in REGγ +/− and REGγ −/− MEFs before and after infection with retrovirus expressing K-ras. C. REGγ −/− MEFs were infected with retrovirus expressing REGγ. Half-lives of p19^Arf were measured as in Figure 2. D. REGγ +/− and REGγ −/− MEFs were infected with retrovirus expressing K-ras. Half-lives of p19^Arf were measured as in Figure 2.