Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 i s regulated by the ubiquitin-proteasome system. Activation of p27 degradati on is seen in proliferating cells and in many types of aggressive human car cinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/C dk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether thr eonine 187 phosphorylation stimulates p27 degradation through the ubiquitin -proteasome system or an alternative pathway is still not known. Here, we d emonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro r econstituted system) is cell-cycle regulated and that Cdk activity is requi red for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G(1)-enriched extracts o nly upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreoni ne 187 site-specific antibody for p27, we show that threonine 187 phosphory lation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G(1) cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires format ion of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable c omplex with it, is unable to stimulate p27 ubiquitination by G(1) extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by c yclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquiti nation. Thus throughout the cell cycle, both phosphorylation and trimeric c omplex formation act as signals for the ubiquitination of a Cdk inhibitor.