MPF localization is controlled by nuclear export

Abstract

In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase.

p21-Mediated Nuclear Retention of Cyclin B1-Cdk1 in Response to Genotoxic Stress

“…The activity of cyclin B1-Cdk1, which plays a crucial role in controlling the initiation and progression of mitosis, is tightly regulated at multiple levels. In cycling cells, cyclin B1-Cdk1 complexes are kept inactive during S and G2 phases by inhibitory phosphorylations on Cdk1 at Thr14 and Tyr15 (by Myt1 and Wee1 kinases), and they are retained in the cytoplasm due to an active nuclear export (Hagting et al, 1998;Yang et al, 1998). The onset of prophase correlates with dephosphorylation of Cdk1 by Cdc25 family phosphatases, phosphorylation of cyclin B1, and a rapid nuclear accumulation of active cyclin B1-Cdk1 (rev.…”

“…In cycling cells, cyclin B1 synthesis increases during late S-early G2 phase (Pines and Hunter, 1989), but the newly formed cyclin B1-Cdk1 complexes are kept inactive by inhibitory phosphorylations on Thr 14 and Tyr 15 (Norbury et al, 1991) and by active and constant nuclear exclusion via Crm1-mediated nuclear export (Hagting et al, 1998;Yang et al, 1998). The rapid nuclear accumulation of cyclin B1-Cdk1 that occurs in prophase coincides with phosphorylation of cyclin B1 at the cytoplasmic retention sequence, which also contains its nuclear export sequence (Hagting et al, 1999; for review, see Porter and Donoghue, 2003).…”

Caffeine and human DNA metabolism: the magic and the mystery

“…Subsequent studies demonstrated that cyclin B1/Cdk1 complexes were also regulated by cellular compartmentalization, with the Crm1 nuclear exporter actively sequestering cyclin B1 in the cytoplasm [57,58]. Phosphorylation of cyclin B1 by the Polo-like kinase, Plk1, blocks Crm1-mediated nuclear export allowing nuclear accumulation of cyclin B1 [59].…”

Negative autoregulation of Oct3/4 through Cdx1 promotes the onset of gastrulation

“…To study the function of Cdx1 as a negative regulator of cell proliferation at the onset of gastrulation, we took advantage of the cyclin B/GFP chimeric protein (Hagting et al, 1998). This fusion protein was shown to exhibit the same cellular localization and degradation at the end of anaphase characteristic of the endogenous cyclin B (Pines and Hunter, 1991;Hagting et al, 1998). The cyclin B/GFP expression vector was coinjected radially at the 2-4-cell stage with Cdx1MO and the fluorescence was monitored at the onset of gastrulation.…”