A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase

Abstract

The protein p27Kip1 is an inhibitor of cell division. An increase in p27 causes proliferating cells to exit from the cell cycle, and a decrease in p27 is necessary for quiescent cells to resume division. Abnormally low amounts of p27 are associated with pathological states of excessive cell proliferation, especially cancers. In normal and tumour cells, p27 is regulated primarily at the level of translation and protein turnover. Phosphorylation of p27 on threonine 187 (T187) by cyclin-dependent kinase 2 (Cdk2) is thought to initiate the major pathway for p27 proteolysis. To critically test the importance of this pathway in vivo, we replaced the murine p27 gene with one that encoded alanine instead of threonine at position 187 (p27T187A). Here we show that cells expressing p27T187A were unable to downregulate p27 during the S and G2 phases of the cell cycle, but that this had a surprisingly modest effect on cell proliferation both in vitro and in vivo. Our efforts to explain this unexpected result led to the discovery of a second proteolytic pathway for controlling p27, one that is activated by mitogens and degrades p27 exclusively during G1.

Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

“…Our results show multiple modes of p27 Kip1 regulation consistent with others' recent studies (Malek et al, 2001;Mirza et al, 2004). One mechanism whereby B-RAF regulates p27 Kip1 is control of mRNA level.…”

Deregulated degradation of the cdk inhibitor p27 and malignant transformation

“…As detailed in the text, p27 and Skp2 function upstream of cyclin E. Due to the nature of these proteins, the G1/S transition is regulated by the alternating activity of proto-oncogenes and tumor suppressors to keep in check the events leading to cell proliferation. event is still dependent on Skp2 [27], the other one favors a model in which p27 is degraded in G1 and perhaps in G0 in a Skp2-independent manner [28]. However, these investigations do not clarify whether the rate of p27 degradation in early-mid G1 is increased compared to G0 or whether translational mechanisms are responsible for p27 reduction in early-mid G1 [29].…”

CRM1/Ran-Mediated Nuclear Export of p27Kip1Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

“…T187 phosphorylation allows recognition of p27 by its SCF-type E3 ligase, comprised of Skp1, Cul1, and the F-box protein, Skp2 and Roc1 and the Cks1 cofactor Ohta et al, 1999;Sutterluty et al, 1999;Tsvetkov et al, 1999;Ganoth et al, 2001;Spruck et al, 2001). Recent evidence suggests that p27 proteolysis is regulated by at least two distinct mechanisms, with mitogenic signaling conditioning p27 for degradation in early G1 in a manner independent of T187 phosphorylation (Hara et al, 2001;Malek et al, 2001), whereas Skp2-dependent cyclin E-Cdk 2-mediated degradation occurs in S phase after T187 phosphorylation (Malek et al, 2001). …”