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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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Nuclear import of GST-cyclin E and GST-cyclin B1  (121–397) is blocked by peptides that interfere with importin- mediated transport. Uptake assays into digitonin-permeabilized  cells were performed for fluorescein maleimide–labeled GST-NLS, GST-IBB, GST-cyclin E, and GST-cyclin B1 (121–397),  and FLUOS-labeled GST-M9 proteins. Assays contained egg cytosol (4 mg/ml final protein concentration), an energy-regenerating system, and either no peptide or one of the following peptides  at a final concentration of 50 μM: NP-NLS (the 20–amino acid  peptide containing the basic bipartite NLS of nucleoplasmin);  IBB, residues 1–64 of importin-α produced by thrombin cleavage  of GST-IBB; or M9, the transportin-dependent NLS of human  hnRNPA1.
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Figure 2: Nuclear import of GST-cyclin E and GST-cyclin B1 (121–397) is blocked by peptides that interfere with importin- mediated transport. Uptake assays into digitonin-permeabilized cells were performed for fluorescein maleimide–labeled GST-NLS, GST-IBB, GST-cyclin E, and GST-cyclin B1 (121–397), and FLUOS-labeled GST-M9 proteins. Assays contained egg cytosol (4 mg/ml final protein concentration), an energy-regenerating system, and either no peptide or one of the following peptides at a final concentration of 50 μM: NP-NLS (the 20–amino acid peptide containing the basic bipartite NLS of nucleoplasmin); IBB, residues 1–64 of importin-α produced by thrombin cleavage of GST-IBB; or M9, the transportin-dependent NLS of human hnRNPA1.

Mentions: To investigate the possible involvement of known soluble transport factors in cyclin import, we preincubated the interphase egg cytosol with competitor peptides designed to prevent importins or transportin from interacting with their cargo. When the interphase egg cytosol used in the digitonin-permeabilized cell import assay was preincubated with peptide encoding the classical NLS from nucleoplasmin (50 μM), fluoresceinated cyclin B (121–397), cyclin E, and control GST-NLS proteins were all prevented from entering nuclei (Fig. 2). These data suggested that cyclins might use the well-known importin-α/β pathway for nuclear import. However, when we used a peptide encoding the IBB domain of importin-α (importin-α residues 1–64) in similar experiments, we found that import of cyclin E, but not cyclin B1 (121–397), was impaired. A peptide encoding the M9 NLS blocked import of only GST-M9 protein and did not interfere with cyclin import. These data suggest that nuclear import of both cyclins E and B1 proceed by importin-dependent, transportin-independent pathways. However, the fact that GST-NLS and GST-IBB proteins interfered differentially with import of cyclins E and B1 suggested, again, that these cyclins might use different 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)

Nuclear import of GST-cyclin E and GST-cyclin B1  (121–397) is blocked by peptides that interfere with importin- mediated transport. Uptake assays into digitonin-permeabilized  cells were performed for fluorescein maleimide–labeled GST-NLS, GST-IBB, GST-cyclin E, and GST-cyclin B1 (121–397),  and FLUOS-labeled GST-M9 proteins. Assays contained egg cytosol (4 mg/ml final protein concentration), an energy-regenerating system, and either no peptide or one of the following peptides  at a final concentration of 50 μM: NP-NLS (the 20–amino acid  peptide containing the basic bipartite NLS of nucleoplasmin);  IBB, residues 1–64 of importin-α produced by thrombin cleavage  of GST-IBB; or M9, the transportin-dependent NLS of human  hnRNPA1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132890&req=5

Figure 2: Nuclear import of GST-cyclin E and GST-cyclin B1 (121–397) is blocked by peptides that interfere with importin- mediated transport. Uptake assays into digitonin-permeabilized cells were performed for fluorescein maleimide–labeled GST-NLS, GST-IBB, GST-cyclin E, and GST-cyclin B1 (121–397), and FLUOS-labeled GST-M9 proteins. Assays contained egg cytosol (4 mg/ml final protein concentration), an energy-regenerating system, and either no peptide or one of the following peptides at a final concentration of 50 μM: NP-NLS (the 20–amino acid peptide containing the basic bipartite NLS of nucleoplasmin); IBB, residues 1–64 of importin-α produced by thrombin cleavage of GST-IBB; or M9, the transportin-dependent NLS of human hnRNPA1.
Mentions: To investigate the possible involvement of known soluble transport factors in cyclin import, we preincubated the interphase egg cytosol with competitor peptides designed to prevent importins or transportin from interacting with their cargo. When the interphase egg cytosol used in the digitonin-permeabilized cell import assay was preincubated with peptide encoding the classical NLS from nucleoplasmin (50 μM), fluoresceinated cyclin B (121–397), cyclin E, and control GST-NLS proteins were all prevented from entering nuclei (Fig. 2). These data suggested that cyclins might use the well-known importin-α/β pathway for nuclear import. However, when we used a peptide encoding the IBB domain of importin-α (importin-α residues 1–64) in similar experiments, we found that import of cyclin E, but not cyclin B1 (121–397), was impaired. A peptide encoding the M9 NLS blocked import of only GST-M9 protein and did not interfere with cyclin import. These data suggest that nuclear import of both cyclins E and B1 proceed by importin-dependent, transportin-independent pathways. However, the fact that GST-NLS and GST-IBB proteins interfered differentially with import of cyclins E and B1 suggested, again, that these cyclins might use different 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.

Show MeSH
Related in: MedlinePlus