Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal.
BACKGROUND
At M phase, cyclin B1 is phosphorylated in the cytoplasmic retention sequence (CRS), which is required for nuclear export. During interphase, cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export. In M phase, cyclin B moves rapidly into the nucleus coincident with its phosphorylation, an overall movement that might be caused simply by a decrease in its nuclear export. However, the questions of whether CRS phosphorylation is required for cyclin B1 translocation in mitosis and whether a reduction in nuclear export is sufficient to explain its rapid relocalisation have not been addressed.


RESULTS
We have used two forms of green fluorescent protein to analyse simultaneously the translocation of wild-type cyclin B1 and a phosphorylation mutant of cyclin B1 in mitosis, and correlated this with an in vitro nuclear import assay. We show that cyclin B1 rapidly translocates into the nucleus approximately 10 minutes before breakdown of the nuclear envelope, and that this movement requires the CRS phosphorylation sites. A cyclin B1 mutant that cannot be phosphorylated enters the nucleus after the wild-type protein. Phosphorylation of the CRS creates a nuclear import signal that enhances cyclin B1 import in vitro and in vivo, in a manner distinct from the previously described import of cyclin B1 mediated by importin beta.


CONCLUSIONS
We show that phosphorylation of human cyclin B1 is required for its rapid translocation to the nucleus towards the end of prophase. Phosphorylation enhances cyclin B1 nuclear import by creating a nuclear import signal. The phosphorylation of the CRS is therefore a critical step in the control of mitosis.
Wellcome/CRC Institute, Department of Zoology, Tennis Court Road, Cambridge, CB2 1QR, UK.
A
A
Hagting
M
M
Jackman
K
K
Simpson
J
J
Pines
D000595:Amino Acid Sequence; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism*; D056744:Cyclin B1; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011418:Prophase / Q000502:physiology*; D016415:Sequence Alignment; D013997:Time Factors
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D008565:Membrane Proteins; C091632:cell-surface retention-binding protein 1