Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus |
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Authors: | There`se David-Pfeuty Yolande Nouvian-Dooghe Dany Rouillard |
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Institution: | The´re`se David-Pfeuty,Yolande Nouvian-Dooghe,Dany Rouillard |
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Abstract: | An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over. |
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Keywords: | cell cycle regulation S-to-M coupling cell cycle protein localization |
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