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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, but not GST-cyclin B1  (121–397), requires importin-α. (A) Ni2+NTA-agarose can be used  to deplete importin-α from egg cytosol. Anti–importin-α Western  blot for mock (Sepharose CL-4B)-depleted (lane 1), Ni2+NTA-agarose–depleted (lane 2), and Ni2+NTA-agarose–depleted extract to which excess recombinant importin-α (100 μg/ml) had  been added back (lane 3). (B) Uptake assays into digitonin-permeabilized cells were performed for fluorescein maleimide–labeled  GST-cyclin E and GST-cyclin B1 (121–397) with mock-depleted  extract (passed over Sepharose CL-6B beads) at both room temperature and on ice, and at room temperature with Ni2+NTA-agarose–depleted extract. All assays contained the same final protein  concentration of extract (4 mg/ml). Recombinant importin-α, 50  μg/ml, was added back to a set of assays performed with  Ni+2NTA-agarose–depleted extract to show that the effect of depletion on import was due primarily to the removal of importin-α.
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Figure 3: Nuclear import of GST-cyclin E, but not GST-cyclin B1 (121–397), requires importin-α. (A) Ni2+NTA-agarose can be used to deplete importin-α from egg cytosol. Anti–importin-α Western blot for mock (Sepharose CL-4B)-depleted (lane 1), Ni2+NTA-agarose–depleted (lane 2), and Ni2+NTA-agarose–depleted extract to which excess recombinant importin-α (100 μg/ml) had been added back (lane 3). (B) Uptake assays into digitonin-permeabilized cells were performed for fluorescein maleimide–labeled GST-cyclin E and GST-cyclin B1 (121–397) with mock-depleted extract (passed over Sepharose CL-6B beads) at both room temperature and on ice, and at room temperature with Ni2+NTA-agarose–depleted extract. All assays contained the same final protein concentration of extract (4 mg/ml). Recombinant importin-α, 50 μg/ml, was added back to a set of assays performed with Ni+2NTA-agarose–depleted extract to show that the effect of depletion on import was due primarily to the removal of importin-α.

Mentions: Since the basic NLS peptide blocked cyclin import, we wished to investigate whether importin-α was required for nuclear import of either cyclin B1 or cyclin E. For this purpose, nuclear import assays were performed in which the egg cytosol was replaced with cytosol depleted of importin-α (by passage through a column of Ni-NTA-agarose; see Görlich et al., 1994) (Fig. 3 A) or with control cytosol mock-depleted with Sepharose CL-6B. GST-cyclin E import was abolished in Ni2+NTA-agarose–treated extracts, whereas cyclin B1 (121–397) import was not severely affected (Fig. 3 B). Readdition of recombinant importin-α to Ni2+NTA-agarose–depleted extracts restored GST-cyclin E nuclear import, though not quite to the levels observed in the mock-depleted extract. These data indicate that cyclin E nuclear import proceeds via an importin-α–dependent mechanism, whereas, surprisingly, given its inhibition by NLS peptide, cyclin B1 import does not.


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, but not GST-cyclin B1  (121–397), requires importin-α. (A) Ni2+NTA-agarose can be used  to deplete importin-α from egg cytosol. Anti–importin-α Western  blot for mock (Sepharose CL-4B)-depleted (lane 1), Ni2+NTA-agarose–depleted (lane 2), and Ni2+NTA-agarose–depleted extract to which excess recombinant importin-α (100 μg/ml) had  been added back (lane 3). (B) Uptake assays into digitonin-permeabilized cells were performed for fluorescein maleimide–labeled  GST-cyclin E and GST-cyclin B1 (121–397) with mock-depleted  extract (passed over Sepharose CL-6B beads) at both room temperature and on ice, and at room temperature with Ni2+NTA-agarose–depleted extract. All assays contained the same final protein  concentration of extract (4 mg/ml). Recombinant importin-α, 50  μg/ml, was added back to a set of assays performed with  Ni+2NTA-agarose–depleted extract to show that the effect of depletion on import was due primarily to the removal of importin-α.
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Related In: Results  -  Collection

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Figure 3: Nuclear import of GST-cyclin E, but not GST-cyclin B1 (121–397), requires importin-α. (A) Ni2+NTA-agarose can be used to deplete importin-α from egg cytosol. Anti–importin-α Western blot for mock (Sepharose CL-4B)-depleted (lane 1), Ni2+NTA-agarose–depleted (lane 2), and Ni2+NTA-agarose–depleted extract to which excess recombinant importin-α (100 μg/ml) had been added back (lane 3). (B) Uptake assays into digitonin-permeabilized cells were performed for fluorescein maleimide–labeled GST-cyclin E and GST-cyclin B1 (121–397) with mock-depleted extract (passed over Sepharose CL-6B beads) at both room temperature and on ice, and at room temperature with Ni2+NTA-agarose–depleted extract. All assays contained the same final protein concentration of extract (4 mg/ml). Recombinant importin-α, 50 μg/ml, was added back to a set of assays performed with Ni+2NTA-agarose–depleted extract to show that the effect of depletion on import was due primarily to the removal of importin-α.
Mentions: Since the basic NLS peptide blocked cyclin import, we wished to investigate whether importin-α was required for nuclear import of either cyclin B1 or cyclin E. For this purpose, nuclear import assays were performed in which the egg cytosol was replaced with cytosol depleted of importin-α (by passage through a column of Ni-NTA-agarose; see Görlich et al., 1994) (Fig. 3 A) or with control cytosol mock-depleted with Sepharose CL-6B. GST-cyclin E import was abolished in Ni2+NTA-agarose–treated extracts, whereas cyclin B1 (121–397) import was not severely affected (Fig. 3 B). Readdition of recombinant importin-α to Ni2+NTA-agarose–depleted extracts restored GST-cyclin E nuclear import, though not quite to the levels observed in the mock-depleted extract. These data indicate that cyclin E nuclear import proceeds via an importin-α–dependent mechanism, whereas, surprisingly, given its inhibition by NLS peptide, cyclin B1 import does not.

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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