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.
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.
David-Pfeuty
T
T
UMR 146 du CNRS, Institut Curie-Recherche, Centre Universitaire, Orsay, France.
Nouvian-Dooghe
Y
Y
Rouillard
D
D
D016475:3T3 Cells; D000818:Animals; D016590:Aphidicolin / Q000378:metabolism; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D003593:Cytoplasm / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D007399:Interphase; D051379:Mice; D008938:Mitosis; D016196:S Phase; D013347:Subcellular Fractions
C534700:Ccnb1 protein, mouse; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D016590:Aphidicolin; D016203:CDC2 Protein Kinase