Ras promotes p21Waf1/Cip1 protein stability via a cyclin D1-imposed block in proteasome-mediated degradation

Ras promotes the accumulation of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Previous studies reported that acute Raf/MEK/ERK activation elevates p21 protein levels by increased transcription. However, we have found that p21 induction in Ras-transformed murine fibroblasts occurs principally by a post-translational mechanism. Chronic activation of the Raf/MEK/ERK pathway blocked proteasome-mediated p21 degradation, resulting in accumulation of p21 protein with an elevated half-life. The stabilization of p21 by Ras was accompanied by high levels of p21-associated cyclin D1 and, similarly to Ras, cyclin D1 was sufficient to inhibit the proteasome-mediated p21 degradation. Knock-down of cyclin D1 by RNA interference confirmed that Ras-induced p21 stabilization was dependent upon cyclin D1 expression. We show that p21 directly binds to the C8alpha subunit of the 20S proteasome complex and that by competing for binding, cyclin D1 inhibits p21 degradation by purified 20S complexes in vitro. Therefore, we propose that Ras stabilizes p21 by promoting the formation of p21-cyclin D1 complexes that prevent p21 association with, and subsequent degradation by, the 20S proteasome.