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

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 alpha -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 affinity between C8 and p21(WAF1/CIP1) significantly reduced the stability of the latter. These data suggest that interaction with a 20S proteasome alpha -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 purified 20S proteasomes in a manner that was dependent on the C8-interaction domain.