Investigation into the molecular mechanisms underlying proteolysis of CDK inhibitor p27

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We have studied the mechamism underlying the regulation of the abundance of the CDK inhibitor p27. p27 is degraded at the GO-G1 transition, and its abundance remains low during cell proliferation. The SCF/Skp2 ubiquitin ligase complex has been thought to play a critical role in p27 degradation. However, analysis of Skp2-deficient mice revealed that degradation of p27 at the GO-G1 transition proceeds normally in Skp2-/-cells, whereas p27 proteolysis during S-G2 phases is impaired in these Skp2-deficient cells. These results suggested the presence of Skp2-independent pathway to control ubiquitination of p27 at the GO-G1 transition. Using in vitro ubiquitylation assay, we biochemically purified a complex consisting of two proteins, designated KPC (Kip1 ubiquitylation Promoting Complex)1 and KPC2. Given that KPC1/2 are localized in the cytoplasm, nuclear export of p27 seems to precede the ubiquitination by KPC1/2. Overexpression of wild-type KPC1/2 in mammalian cells promotes the degradation of p27, whereas expression of dominant-negative mutant KPC 1/2 delayed the degradation. Depletion of KPC1 by RNA interference also inhibited p27 degradation. These data suggest that KPC 1/2 ubiquitin ligase complex controls the degradation of p27 at the GO-G1 transition, whereas the major function of Skp2 may be the regulation of progression from G2 to M phase by mediating the degradation of p27.