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      Activation of cyclin B1–Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis

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      The Journal of Cell Biology
      The Rockefeller University Press

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          Abstract

          A biosensor for cyclin B–Cdk1 activity shows that it uses an unconventional yet simple mechanism for nuclear accumulation.

          Abstract

          The cyclin B–Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for cyclin B1–Cdk1 activity to show that activating cyclin B1–Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of cyclin B1–Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between cyclin B1–Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry.

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          Most cited references56

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          Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.

          CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G(2)/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.
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            An improved cyan fluorescent protein variant useful for FRET.

            Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.
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              Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.

              We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted PBD-substrate binding and localization of the PBD to centrosomes. This finding reveals how Plk1 can localize to specific sites within cells in response to Cdk phosphorylation at those sites and provides a structural mechanism for targeting the Plk1 kinase domain to its substrates.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                19 April 2010
                : 189
                : 2
                : 247-259
                Affiliations
                The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, England, UK
                Author notes
                Correspondence to Olivier Gavet: olivier.gavet@ 123456gurdon.cam.ac.uk ; or Jonathon Pines: jp103@ 123456cam.ac.uk

                O. Gavet's present address is Université Pierre et Marie Curie, 75252 Paris, Cedex 05, France.

                Article
                200909144
                10.1083/jcb.200909144
                2856909
                20404109
                e811ab08-549c-47e8-a4cd-eb0be5a450e8
                © 2010 Gavet and Pines

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                : 29 September 2009
                : 22 March 2010
                Categories
                Research Articles
                Article

                Cell biology
                Cell biology

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