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 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 vitro 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.