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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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(A) Cyclin B1 (121–397), but not importin-α, is capable  of binding to an importin-β truncation mutant containing residues 1–462. Interphase egg cytosol, diluted 1:3 with PAT buffer,  was supplemented with 1/50 vol of reticulocyte lysate containing  radiolabeled cyclins E and B1 (121–397) and incubated at 4°C  with glutathione-Sepharose beads containing immobilized GST,  GST-importin-β (full-length), or GST-importin-β (1–462) for 40  min. The beads were recovered by centrifugation and after extensive washing, bound proteins were eluted and separated by SDS-PAGE (along with 1/10 vol of the cyclin-extract mixture that had  not been incubated with beads). Western blots were probed for  endogenous importin-α, while radiolabeled cyclins E and B1  (121–397) were detected by autoradiography of dried gels. (B)  Evidence that cyclin B1 and the IBB domain of importin-α can  bind simultaneously to importin-β. The same cyclin E and B1  (121–397)–supplemented egg cytosol as used above was incubated  with glutathione-Sepharose beads coupled to GST, GST-NLS,  GST-IBB, or GST-IBB55 fusion proteins. Bead-bound proteins  were recovered and separated (along with a control representing  10% of the input protein), as before. Western blots were probed  with antibodies against importin-α and -β to detect endogenous  proteins bound to the beads; the radiolabeled cyclins were detected by autoradiography.
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Figure 6: (A) Cyclin B1 (121–397), but not importin-α, is capable of binding to an importin-β truncation mutant containing residues 1–462. Interphase egg cytosol, diluted 1:3 with PAT buffer, was supplemented with 1/50 vol of reticulocyte lysate containing radiolabeled cyclins E and B1 (121–397) and incubated at 4°C with glutathione-Sepharose beads containing immobilized GST, GST-importin-β (full-length), or GST-importin-β (1–462) for 40 min. The beads were recovered by centrifugation and after extensive washing, bound proteins were eluted and separated by SDS-PAGE (along with 1/10 vol of the cyclin-extract mixture that had not been incubated with beads). Western blots were probed for endogenous importin-α, while radiolabeled cyclins E and B1 (121–397) were detected by autoradiography of dried gels. (B) Evidence that cyclin B1 and the IBB domain of importin-α can bind simultaneously to importin-β. The same cyclin E and B1 (121–397)–supplemented egg cytosol as used above was incubated with glutathione-Sepharose beads coupled to GST, GST-NLS, GST-IBB, or GST-IBB55 fusion proteins. Bead-bound proteins were recovered and separated (along with a control representing 10% of the input protein), as before. Western blots were probed with antibodies against importin-α and -β to detect endogenous proteins bound to the beads; the radiolabeled cyclins were detected by autoradiography.

Mentions: The most direct way to test the hypothesis that cyclin B1 and importin-α bound to different sites on importin-β was to identify an importin-β mutant that could bind to one protein, but not the other. Kutay et al. (1997) have shown that the IBB domain of importin-α requires residues 286– 876 of importin-β for binding, while Ran-GTP and certain nucleoporins bind to the NH2-terminal half (residues 1–462) of the importin-β protein. As shown in Fig. 6 A, cyclin B1 bound to both full-length and 1–462 importin-β. Consistent with a requirement for importin-α in the cyclin E–importin-β interaction, the 1–462 mutant could bind neither importin-α from the egg cytosol nor cyclin E (Fig. 6 A). These data confirm that cyclin B1 binds to a different site on importin-β from that used by the IBB domain of importin-α. Very recently, Jäkel and Görlich (1998) identified an NLS in ribosomal protein L23 that binds in a Ran-GTP–sensitive manner to the first 462 amino acids of importin-β, indicating that such interactions can be relevant to nuclear transport processes.


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)

(A) Cyclin B1 (121–397), but not importin-α, is capable  of binding to an importin-β truncation mutant containing residues 1–462. Interphase egg cytosol, diluted 1:3 with PAT buffer,  was supplemented with 1/50 vol of reticulocyte lysate containing  radiolabeled cyclins E and B1 (121–397) and incubated at 4°C  with glutathione-Sepharose beads containing immobilized GST,  GST-importin-β (full-length), or GST-importin-β (1–462) for 40  min. The beads were recovered by centrifugation and after extensive washing, bound proteins were eluted and separated by SDS-PAGE (along with 1/10 vol of the cyclin-extract mixture that had  not been incubated with beads). Western blots were probed for  endogenous importin-α, while radiolabeled cyclins E and B1  (121–397) were detected by autoradiography of dried gels. (B)  Evidence that cyclin B1 and the IBB domain of importin-α can  bind simultaneously to importin-β. The same cyclin E and B1  (121–397)–supplemented egg cytosol as used above was incubated  with glutathione-Sepharose beads coupled to GST, GST-NLS,  GST-IBB, or GST-IBB55 fusion proteins. Bead-bound proteins  were recovered and separated (along with a control representing  10% of the input protein), as before. Western blots were probed  with antibodies against importin-α and -β to detect endogenous  proteins bound to the beads; the radiolabeled cyclins were detected by autoradiography.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132890&req=5

Figure 6: (A) Cyclin B1 (121–397), but not importin-α, is capable of binding to an importin-β truncation mutant containing residues 1–462. Interphase egg cytosol, diluted 1:3 with PAT buffer, was supplemented with 1/50 vol of reticulocyte lysate containing radiolabeled cyclins E and B1 (121–397) and incubated at 4°C with glutathione-Sepharose beads containing immobilized GST, GST-importin-β (full-length), or GST-importin-β (1–462) for 40 min. The beads were recovered by centrifugation and after extensive washing, bound proteins were eluted and separated by SDS-PAGE (along with 1/10 vol of the cyclin-extract mixture that had not been incubated with beads). Western blots were probed for endogenous importin-α, while radiolabeled cyclins E and B1 (121–397) were detected by autoradiography of dried gels. (B) Evidence that cyclin B1 and the IBB domain of importin-α can bind simultaneously to importin-β. The same cyclin E and B1 (121–397)–supplemented egg cytosol as used above was incubated with glutathione-Sepharose beads coupled to GST, GST-NLS, GST-IBB, or GST-IBB55 fusion proteins. Bead-bound proteins were recovered and separated (along with a control representing 10% of the input protein), as before. Western blots were probed with antibodies against importin-α and -β to detect endogenous proteins bound to the beads; the radiolabeled cyclins were detected by autoradiography.
Mentions: The most direct way to test the hypothesis that cyclin B1 and importin-α bound to different sites on importin-β was to identify an importin-β mutant that could bind to one protein, but not the other. Kutay et al. (1997) have shown that the IBB domain of importin-α requires residues 286– 876 of importin-β for binding, while Ran-GTP and certain nucleoporins bind to the NH2-terminal half (residues 1–462) of the importin-β protein. As shown in Fig. 6 A, cyclin B1 bound to both full-length and 1–462 importin-β. Consistent with a requirement for importin-α in the cyclin E–importin-β interaction, the 1–462 mutant could bind neither importin-α from the egg cytosol nor cyclin E (Fig. 6 A). These data confirm that cyclin B1 binds to a different site on importin-β from that used by the IBB domain of importin-α. Very recently, Jäkel and Görlich (1998) identified an NLS in ribosomal protein L23 that binds in a Ran-GTP–sensitive manner to the first 462 amino acids of importin-β, indicating that such interactions can be relevant to nuclear transport processes.

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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