Biological activity of p27^kip1 and its amino- and carboxy-terminal domains in G2/M transition of Xenopus oocytes

Abstract

p27^kip1 is a general inhibitor of Cdks that preferentially accumulates and functions during G1 phase, before the restriction point of the mammalian cell cycle. We observed that injection of purified p27^kip1 into Xenopus oocytes potently inhibits the G2/M transition and activation/dephosphorylation of the maturation promoting factor (MPF, p34^cdc2/cyclin B complex) kinase associated with germinal vesicle breakdown (GVBD) induced by progesterone or insulin. Addition of exogenous p27^kip1 in vitro to lysates of hormonally matured oocytes blocked the enzymatic activity of the activated MPF kinase present in those extracts. Interestingly, the isolated amino-terminal region of p27^kip1 (p27N), encompassing only the Cdk binding site, exhibited a similar inhibitory behavior in vitro and a weaker inhibitory effect in vivo than the complete p27^kip1 protein. Surprisingly, the remaining carboxy-terminal region of p27^kip1 (p27C) actually induced GVBD when injected alone into the oocytes, and also accelerated the kinetics of insulin- or progesterone-induced GVBD. Consistent with the in vivo observations, p27C formed a complex with, and activated, the MPF kinase in lysates of immature oocytes, although this activation was blocked by simultaneous addition of p27N or complete p27^kip1. Active MPF was able to phosphorylate p27C only in the absence of p27N or whole p27^kip1, suggesting that the inhibitory activity associated with the amino terminus is dominant over the activation produced by p27C. These results demonstrate the functional interaction of p27^kip1 with cyclin B/p34^cdc2 complexes during G2/M progression in oocytes, and suggest that the amino and carboxy terminal portions of this protein may play opposite regulatory roles, reminiscent of the corresponding N- and C-terminal portions of p21^waf. We speculate that accumulation of a truncated, C-terminal p27 fragment may play a physiological regulatory role in progression through G2 and later stages of the cell cycle.