Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. in vivo. Conversely, knockdown of MDMX induced the level of endogenous p21 proteins that no longer cofractionated with 26S proteasome, resulting in G1 arrest. The level of p21 was low at early S phase but markedly induced by knocking down either MDMX or MDM2 in human cells. Ablation of p21 rescued the G1 arrest caused by double depletion of MDM2 and MDMX in p53-null cells. These results demonstrate that MDMX and MDM2 independently and cooperatively regulate the proteasome-mediated degradation of p21 at the G1 and early S phases. Proteolysis maintains normal homeostasis of cell cycle-regulated proteins and is crucial for the progression of the cell cycle. These proteins Rabbit polyclonal to HERC4 include cyclins and cyclin-dependent kinase inhibitors, such as p27 or p21 (9, 45, 51). Proteolytic pathways have been recognized and well characterized for many of these cell cycle proteins (34, 36, 38). For instance, ubiquitylation and turnover of cyclin E are implemented by the SCFFbw7 complex during the cell cycle (23, 54). Also, it has been exhibited that p27 degradation is usually facilitated by the SCFSkp2 complex-mediated ubiquitylation and ubiquitin-dependent proteasomal system (6, 50). In contrast, the molecular mechanisms by which p21 is MG-101 usually degraded during the cell cycle are still under argument (4, 8, 21, 44). While it is usually indisputable that p21 is usually degraded through the proteasome system (3, 12), argument still remains as to whether p21 degradation is usually mediated through a ubiquitin-dependent or -impartial proteasome system (4, 8, 21, 44). It is obvious that p21 is usually ubiquitylated in cells (30, 35). Several studies have suggested that Skp2 may target p21’s N terminus for ubiquitylation and p21 degradation (4, 5, 10, 53). However, these notions have been challenged by other studies (2, 7, 21, 28, 44), showing that p21 ubiquitylation is usually dispensable for its proteasomal degradation in vitro and in vivo (19, 29, 44). Also, the N terminus of p21 was shown to be acetylated and therefore less likely to be ubiquitylated (7). In addition, knocking out experienced no effect on p21 turnover (7). Recently, we found that UV-induced p21 turnover was also impartial of ubiquitin and Skp2 (27). Hence, it appears that Skp2 would not be a major player in regulating p21 stability during the cell cycle. So, the question remains which protein(s) regulates p21’s proteasomal degradation during the cell cycle. It is possible that p21 may be degraded directly by the 20S proteasome during the cell cycle, as it has been shown that this 20S proteasome binds to and directly degrades p21 in vitro (29, 49). However, this notion has not been verified in vivo using synchronized cells. Also, one study showed that endogenous p21 cofractionated with the 26S proteasome (43), suggesting that this 26S MG-101 proteasome may participate in the proteolysis of p21. Thus, it is still uncertain which proteasome complex is in charge of p21 turnover during the cell cycle. The other possible candidate would be MDM2 (also called HDM2 in humans; MDM2 used here for simplicity). MDM2 was originally discovered as an E3 MG-101 ubiquitin ligase that specifically mediates p53’s ubiquitylation and degradation (11, 15, 16, 24). Later studies by others and us have shown that MDM2 also mediates p21 degradation by a ubiquitin-independent mechanism (21, 58). However, whether MDM2 exerts this p21 degradation activity during the cell cycle and whether you will find other proteins that may regulate this MDM2 activity remain to be resolved. In our initial experiments to address these questions, we have observed that knocking down the endogenous MDM2 level partially rescued the low level of p21 at early S phase during the cell cycle. In addition, we have explored the possibility of whether MDMX (also called HDMX or MDM4; MDMX used here for simplicity), an MDM2 analog that has been shown to aid MDM2 in suppressing p53 function in cells and animals (18, 25, 33, 39, 41, 46), also works with MDM2 in degrading p21. Indeed, we found that MDMX not only cooperated with MDM2 in degrading p21 in cells but also mediated p21’s degradation.