Biological activity of p27kip1 and its amino- and carboxy-terminal domains in G2/M transition of Xenopus oocytes.
p27kip1 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 p27kip1 into Xenopus oocytes potently inhibits the G2/M transition and activation/dephosphorylation of the maturation promoting factor (MPF, p34cdc2/cyclin B complex) kinase associated with germinal vesicle breakdown (GVBD) induced by progesterone or insulin. Addition of exogenous p27kip1 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 p27kip1 (p27N), encompassing only the Cdk binding site, exhibited a similar inhibitory behavior in vitro and a weaker inhibitory effect in vivo than the complete p27kip1 protein. Surprisingly, the remaining carboxy-terminal region of p27kip1 (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 p27kip1. Active MPF was able to phosphorylate p27C only in the absence of p27N or whole p27kip1, 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 p27kip1 with cyclin B/p34cdc2 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 p21waf. 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.
Font de Mora
J
J
Laboratory of Cellular and Molecular Biology, National Cancer Institute, DBS, Bethesda, Maryland 20892, USA.
Uren
A
A
Heidaran
M
M
Santos
E
E
D000818:Animals; D016203:CDC2 Protein Kinase / Q000502:physiology; D018797:Cell Cycle Proteins; D019926:Cyclin B / Q000502:physiology; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005260:Female; D016195:G2 Phase; D008540:Meiosis; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D008938:Mitosis; D009865:Oocytes / Q000502:physiology; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D019926:Cyclin B; D008869:Microtubule-Associated Proteins; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C493963:Xicl protein, Xenopus; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016203:CDC2 Protein Kinase