Down-regulation of p27Kip1by Two Mechanisms, Ubiquitin-mediated Degradation and Proteolytic Processing

Michiko Shirane, Yumiko Harumiya, Noriko Ishida, Aizan Hirai, Chikara Miyamoto, Shigetsugu Hatakeyama, Kei-ichi Nakayama, Masatoshi Kitagawa
1999 Journal of Biological Chemistry  
The intracellular level of p27 Kip1 , a cyclin-dependent kinase (CDK) inhibitory protein, is rapidly reduced at the G 1 /S transition phase when the cell cycle pause ceases. In this study, we demonstrated that two posttranslational mechanisms were involved in p27 Kip1 breakdown: degradation via the ubiquitin (Ub)-proteasome pathway and proteolytic processing that rapidly eliminates the cyclin-binding domain. We confirmed that p27 Kip1 was ubiquitinated in vitro as well as in vivo. The p27 Kip1
more » ... vivo. The p27 Kip1 -ubiquitination activity was higher at the G 1 /S boundary than during the G 0 /G 1 phase, and p27 Kip1 ubiquitination was reduced significantly when the lysine residues at positions 134, 153, and 165 were replaced by arginine, suggesting that these lysine residues are the targets for Ub conjugation. In parallel with its Ub-dependent degradation, p27 Kip1 was processed rapidly at its N terminus, reducing its molecular mass from 27 to 22 kDa, by a ubiquitination-independent but adenosine triphosphate (ATP)-dependent mechanism with higher activity during the S than the G 0 /G 1 phase. This 22-kDa intermediate had no cyclin-binding domain at its N terminus and virtually no CDK2 kinase inhibitory activity. These results suggest that p27 Kip1 is eliminated by two independent mechanisms, ubiquitin-mediated degradation and ubiquitin-independent processing, during progression from the G 1 to S phase. Cell cycle progression is controlled by a series of kinase complexes composed of cyclins and cyclin-dependent kinases (CDKs) 1 (1). The enzymatic activities of cyclin/CDK complexes are regulated by many mechanisms that reflect both the diver-
doi:10.1074/jbc.274.20.13886 pmid:10318797 fatcat:vt5eiejpjvaplokbzlgnu2jec4