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Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Moore JD, Yang J, Truant R, Kornbluth S - J. Cell Biol. (1999)

Bottom Line: We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component.Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer.In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

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Recombinant importins direct cyclin transport into  nuclei from permeabilized HeLa  cells. Isolated HeLa cell nuclei  from digitonin-permeabilized cells  were incubated in the presence  of recombinant Ran and NTF2  proteins along with one of the  following fluorescently labeled  substrate proteins: GST-cyclin B1  (121–397), GST-cyclin E, GST-SV-40 T antigen NLS, or GST-M9. Assays were supplemented  with recombinant importin-α, importin-β, or both or with MBP-transportin. In control assays,  reticulocyte lysate was used as a  source of all soluble transport  factors. This was chosen rather  than egg cytosol because it had  been demonstrated previously  to support robust import of  transportin cargo in this assay.
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Figure 7: Recombinant importins direct cyclin transport into nuclei from permeabilized HeLa cells. Isolated HeLa cell nuclei from digitonin-permeabilized cells were incubated in the presence of recombinant Ran and NTF2 proteins along with one of the following fluorescently labeled substrate proteins: GST-cyclin B1 (121–397), GST-cyclin E, GST-SV-40 T antigen NLS, or GST-M9. Assays were supplemented with recombinant importin-α, importin-β, or both or with MBP-transportin. In control assays, reticulocyte lysate was used as a source of all soluble transport factors. This was chosen rather than egg cytosol because it had been demonstrated previously to support robust import of transportin cargo in this assay.

Mentions: The binding assay data, peptide competitions, and permeabilized cell assays lead to the conclusion that cyclin B1 is imported into nuclei via direct interactions with importin-β and that cyclin E uses the importin-α/β heterodimer. Moreover, these import reactions are not dependent upon Cdks. As a final, definitive test of these conclusions, we performed nuclear transport assays using digitonin-permeabilized cells and entirely recombinant import factors. All assays contained recombinant Ran and its accessory factor, NTF2. Fluoresceinated GST-cyclin E, like GST fused to the NLS of SV-40 T antigen, was imported into nuclei only in the presence of both recombinant importin-α and -β, while cyclin B1 (121–397) required only importin-β (Fig. 7). The full lysate (here reticulocyte lysate) containing all factors imported both cyclins; recombinant transportin could support nuclear import of only the GST-M9 protein. These results establish that cyclin B1 and cyclin E use distinct importin-mediated pathways for nuclear import.


Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Moore JD, Yang J, Truant R, Kornbluth S - J. Cell Biol. (1999)

Recombinant importins direct cyclin transport into  nuclei from permeabilized HeLa  cells. Isolated HeLa cell nuclei  from digitonin-permeabilized cells  were incubated in the presence  of recombinant Ran and NTF2  proteins along with one of the  following fluorescently labeled  substrate proteins: GST-cyclin B1  (121–397), GST-cyclin E, GST-SV-40 T antigen NLS, or GST-M9. Assays were supplemented  with recombinant importin-α, importin-β, or both or with MBP-transportin. In control assays,  reticulocyte lysate was used as a  source of all soluble transport  factors. This was chosen rather  than egg cytosol because it had  been demonstrated previously  to support robust import of  transportin cargo in this assay.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: Recombinant importins direct cyclin transport into nuclei from permeabilized HeLa cells. Isolated HeLa cell nuclei from digitonin-permeabilized cells were incubated in the presence of recombinant Ran and NTF2 proteins along with one of the following fluorescently labeled substrate proteins: GST-cyclin B1 (121–397), GST-cyclin E, GST-SV-40 T antigen NLS, or GST-M9. Assays were supplemented with recombinant importin-α, importin-β, or both or with MBP-transportin. In control assays, reticulocyte lysate was used as a source of all soluble transport factors. This was chosen rather than egg cytosol because it had been demonstrated previously to support robust import of transportin cargo in this assay.
Mentions: The binding assay data, peptide competitions, and permeabilized cell assays lead to the conclusion that cyclin B1 is imported into nuclei via direct interactions with importin-β and that cyclin E uses the importin-α/β heterodimer. Moreover, these import reactions are not dependent upon Cdks. As a final, definitive test of these conclusions, we performed nuclear transport assays using digitonin-permeabilized cells and entirely recombinant import factors. All assays contained recombinant Ran and its accessory factor, NTF2. Fluoresceinated GST-cyclin E, like GST fused to the NLS of SV-40 T antigen, was imported into nuclei only in the presence of both recombinant importin-α and -β, while cyclin B1 (121–397) required only importin-β (Fig. 7). The full lysate (here reticulocyte lysate) containing all factors imported both cyclins; recombinant transportin could support nuclear import of only the GST-M9 protein. These results establish that cyclin B1 and cyclin E use distinct importin-mediated pathways for nuclear import.

Bottom Line: We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component.Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer.In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

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