A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 alpha-subunit of the 20S proteasome

Abstract

The cyclin-dependent kinase inhibitor p21 WAF1/CIP1 is a key regulator of cell-cycle progression and its expression is tightly regulated at the level of transcription and by proteasome-dependent proteolysis. The turnover of p21 WAF1/CIP1 by proteasomes does not always require the ubiquitylation of p21 WAF1/CIP1 suggesting that there could be an alternative pathway into the proteasome. Here we show that the C8 a-subunit of the 20S proteasome interacts with the C-terminus of p21 WAF1/CIP1 and mediates the degradation of p21 WAF1/CIP1 . A small deletion in this region that disrupts binding to C8 increased the half-life of p21 WAF1/CIP1 expressed in vivo. In contrast a deletion that increased the af®nity between C8 and p21 WAF1/CIP1 signi®cantly reduced the stability of the latter. These data suggest that interaction with a 20S proteasome a-subunit is a critical determinant of p21 WAF1/CIP1 turn-over and show how non-ubiquitylated molecules might bypass the 19S regulator of the proteasome and become targeted directly to the 20S, core protease. Consistent with this, p21 WAF1/CIP1 was degraded rapidly by puri®ed 20S proteasomes in a manner that was dependent on the C8-interaction domain.

The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity

“…The mechanism leading to ERM instability may thus be largely independent of ubiquitination and might involve direct interaction of the C-terminal region of ERM with the proteasome. Such a mechanism has been demonstrated in the case of p21 Cip1 and IkBa, which bind respectively to subunit C8 or C3 of the 20S proteasome (Touitou et al, 2001;Alvarez-Castelao and Castano, 2005). Alternatively, proteasome recognition might involve a cofactor as shown for ODC.…”

An HDAC1-binding domain within FATS bridges p21 turnover to radiation-induced tumorigenesis

“…FATSmediated acetylation of p21 may not only inhibit p21 degradation by proteasome, but also interfere with its phosphorylation by some oncogenic kinase to retain its nuclear localization and proper function to regulate cell cycle. The importance of acetylation modification of p21 is further emphasized by the fact that the C terminal p21 interacts with PCNA, a proliferating cell nuclear antigen (Touitou et al, 2001). The stabilization of p21 by FATS may also facilitate p21-mediated regulation of cell-cycle progression by inhibiting PCNA, independently of CDK inhibition.…”

“…It is noteworthy that the C terminus of p21, namely p21 (139-164), which was bound to both C8 a-subunit of 20S proteasome (Touitou et al, 2001) and REGg proteasome activator (Chen et al, 2007), has an essential role in direct degradation of p21 by proteasome (Sheaff et al, 2000;Chen et al, 2007;Li et al, 2007). In addition, there are four lysine residues (K141, K154, K161 and K163) in the C terminus of p21 protein, which raising the possibility that FATS-mediated acetylation might be directly correlated with p21 stabilization.…”

“…Unbound p21 is directly degraded by proteasome independently of ubiquitination, as the endogenous p21 is fully acetylated at its amino terminus and is therefore not a substrate for N-end rule ubiquitination (Varshavsky et al, 2000). Ubiquitin-independent turnover of p21 results from the interaction of its C terminus with the C8 a-subunit of 20S proteasome (Touitou et al, 2001) or proteasome activator REGg (Chen et al, 2007). In addition, the inhibition of histone deacetylases (HDACs) has been known to selectively induce p21 (Johnstone, 2002;Dokmanovic et al, 2007), although the impact of acetylation on p21 turnover remains uncertain.…”

hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress

“…Instead, p21 was shown to physically interact with the C8 a-subunit of the proteasome and overexpression of mutant forms of p21 unable to bind the proteasome showed greatly enhanced stability. Therefore, proteasomal degradation of p21 can proceed through unique mechanisms including direct binding to the proteasome (Touitou et al, 2001;Coulombe et al, 2004). Since PIKKs are activated when cells are damaged and the proteasome is responsible for degrading proteins, understanding how PIKKs control proteasome function may provide insight into how cells remove damaged proteins when injured.…”