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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) Nuclear import of radiolabeled cyclins in Xenopus  oocytes. The cytoplasm of 12 stage VI oocytes for each time point  was injected with a mixture of 14C-labeled BSA and 35S-labeled in  vitro translated Xenopus cyclin E, A1, B1, or a B1 truncation mutant (residues 121–397) lacking the NES near the NH2 terminus.  Immediately after injection, or after 4, 6, 9, or 18 h of incubation at  18°C, half of the oocytes were manually dissected into nuclear and  cytoplasmic fractions. Samples were processed as described previously (Yang et al., 1998) and fractions representing the total, cytoplasmic, or nuclear fractions of three oocytes were separated by  SDS-PAGE and detected by autoradiography. (B) Cyclin transport into the nuclei of digitonin-permeabilized cells. Import assays  were performed essentially as described in Materials and Methods  with interphase Xenopus egg cytosol diluted 1:10 (4 mg protein/ml  final concentration) to provide a source of soluble transport factors. Fluorescein-labeled GST or GST-fusion proteins [GST-NLS,  GST-cyclin E, and GST-cyclin B1 (121–397)] were added to assays  at final concentrations of 50–100 μg/ml. Roscovitine (20 μM) was  included in the assays to prevent activation of Cdc2/cyclin B1 complexes and consequent nuclear envelope breakdown. Control reactions were performed on ice. At least eight fields were examined  visually for each assay. The degree of nuclear fluorescence showed  little variation from field to field. Identical exposures were taken  for the assays performed on ice and at room temperature.
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Figure 1: (A) Nuclear import of radiolabeled cyclins in Xenopus oocytes. The cytoplasm of 12 stage VI oocytes for each time point was injected with a mixture of 14C-labeled BSA and 35S-labeled in vitro translated Xenopus cyclin E, A1, B1, or a B1 truncation mutant (residues 121–397) lacking the NES near the NH2 terminus. Immediately after injection, or after 4, 6, 9, or 18 h of incubation at 18°C, half of the oocytes were manually dissected into nuclear and cytoplasmic fractions. Samples were processed as described previously (Yang et al., 1998) and fractions representing the total, cytoplasmic, or nuclear fractions of three oocytes were separated by SDS-PAGE and detected by autoradiography. (B) Cyclin transport into the nuclei of digitonin-permeabilized cells. Import assays were performed essentially as described in Materials and Methods with interphase Xenopus egg cytosol diluted 1:10 (4 mg protein/ml final concentration) to provide a source of soluble transport factors. Fluorescein-labeled GST or GST-fusion proteins [GST-NLS, GST-cyclin E, and GST-cyclin B1 (121–397)] were added to assays at final concentrations of 50–100 μg/ml. Roscovitine (20 μM) was included in the assays to prevent activation of Cdc2/cyclin B1 complexes and consequent nuclear envelope breakdown. Control reactions were performed on ice. At least eight fields were examined visually for each assay. The degree of nuclear fluorescence showed little variation from field to field. Identical exposures were taken for the assays performed on ice and at room temperature.

Mentions: The enormous size of the Xenopus oocyte has made it one of the systems of choice for nuclear transport studies. Microinjection of radiolabeled transport cargo into either the nuclear or cytoplasmic compartment, followed by manual oocyte dissection, allows rapid quantitation of nuclear import and export rates. As a starting point for examining the pathways responsible for nuclear import of Cdk/cyclin complexes, we followed the fate of radiolabeled cyclins injected into the cytoplasm of stage VI oocytes. Since Cdks are present in excess in the oocyte, each injected cyclin should bind to its cognate Cdk. Full-length 35S-labeled, in vitro translated Xenopus cyclins E, A1, and B1 were individually injected into the cytoplasm of stage VI oocytes along with 14C-labeled BSA as a control. The nuclear and cytoplasmic fractions were then manually separated immediately, 4 or 9 h after injection. As shown in Fig. 1 A, BSA remained in the cytoplasm for at least 9 h after injection, while cyclins E and A1 translocated to the nucleus. Within 4 h, virtually all of the cyclin E and 50% of the cyclin A1 had translocated to the nucleus; the great majority of cyclin A1 was nuclear by 9 h. In contrast, cyclin B1 injected into the cytoplasm remained there for at least 18 h, consistent with its normally interphase cytoplasmic localization. A truncated form of cyclin B1 protein, which eliminates the NES (cycB1 121–397), entered the nucleus, albeit more slowly than cyclins E and A1; somewhat less than 50% of the injected protein had accumulated in the nucleus 9 h after injection. These data suggest that the nuclear import rates of all three cyclins are different, raising the possibility that they are recognized by the import machinery with different affinities or that distinct import pathways are used. Indeed, when we added fluorescein-conjugated, recombinant GST-cyclins B1 and E to Xenopus egg extracts containing ∼1,000 nuclei/μl, fluorescent cyclin E was clearly transported into nuclei more efficiently than cyclin B1, even after removal of the cyclin B1 NES (data not shown). We selected these cyclins, the fastest and slowest for nuclear import, for further study. Interestingly, these cyclins form complexes with different catalytic subunits. Moreover, in combination, Cdk2/cyclin E and Cdc2/cyclin B1 complexes are fully capable of driving multiple rounds of S phase and M phase in Xenopus egg extracts.


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) Nuclear import of radiolabeled cyclins in Xenopus  oocytes. The cytoplasm of 12 stage VI oocytes for each time point  was injected with a mixture of 14C-labeled BSA and 35S-labeled in  vitro translated Xenopus cyclin E, A1, B1, or a B1 truncation mutant (residues 121–397) lacking the NES near the NH2 terminus.  Immediately after injection, or after 4, 6, 9, or 18 h of incubation at  18°C, half of the oocytes were manually dissected into nuclear and  cytoplasmic fractions. Samples were processed as described previously (Yang et al., 1998) and fractions representing the total, cytoplasmic, or nuclear fractions of three oocytes were separated by  SDS-PAGE and detected by autoradiography. (B) Cyclin transport into the nuclei of digitonin-permeabilized cells. Import assays  were performed essentially as described in Materials and Methods  with interphase Xenopus egg cytosol diluted 1:10 (4 mg protein/ml  final concentration) to provide a source of soluble transport factors. Fluorescein-labeled GST or GST-fusion proteins [GST-NLS,  GST-cyclin E, and GST-cyclin B1 (121–397)] were added to assays  at final concentrations of 50–100 μg/ml. Roscovitine (20 μM) was  included in the assays to prevent activation of Cdc2/cyclin B1 complexes and consequent nuclear envelope breakdown. Control reactions were performed on ice. At least eight fields were examined  visually for each assay. The degree of nuclear fluorescence showed  little variation from field to field. Identical exposures were taken  for the assays performed on ice and at room temperature.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (A) Nuclear import of radiolabeled cyclins in Xenopus oocytes. The cytoplasm of 12 stage VI oocytes for each time point was injected with a mixture of 14C-labeled BSA and 35S-labeled in vitro translated Xenopus cyclin E, A1, B1, or a B1 truncation mutant (residues 121–397) lacking the NES near the NH2 terminus. Immediately after injection, or after 4, 6, 9, or 18 h of incubation at 18°C, half of the oocytes were manually dissected into nuclear and cytoplasmic fractions. Samples were processed as described previously (Yang et al., 1998) and fractions representing the total, cytoplasmic, or nuclear fractions of three oocytes were separated by SDS-PAGE and detected by autoradiography. (B) Cyclin transport into the nuclei of digitonin-permeabilized cells. Import assays were performed essentially as described in Materials and Methods with interphase Xenopus egg cytosol diluted 1:10 (4 mg protein/ml final concentration) to provide a source of soluble transport factors. Fluorescein-labeled GST or GST-fusion proteins [GST-NLS, GST-cyclin E, and GST-cyclin B1 (121–397)] were added to assays at final concentrations of 50–100 μg/ml. Roscovitine (20 μM) was included in the assays to prevent activation of Cdc2/cyclin B1 complexes and consequent nuclear envelope breakdown. Control reactions were performed on ice. At least eight fields were examined visually for each assay. The degree of nuclear fluorescence showed little variation from field to field. Identical exposures were taken for the assays performed on ice and at room temperature.
Mentions: The enormous size of the Xenopus oocyte has made it one of the systems of choice for nuclear transport studies. Microinjection of radiolabeled transport cargo into either the nuclear or cytoplasmic compartment, followed by manual oocyte dissection, allows rapid quantitation of nuclear import and export rates. As a starting point for examining the pathways responsible for nuclear import of Cdk/cyclin complexes, we followed the fate of radiolabeled cyclins injected into the cytoplasm of stage VI oocytes. Since Cdks are present in excess in the oocyte, each injected cyclin should bind to its cognate Cdk. Full-length 35S-labeled, in vitro translated Xenopus cyclins E, A1, and B1 were individually injected into the cytoplasm of stage VI oocytes along with 14C-labeled BSA as a control. The nuclear and cytoplasmic fractions were then manually separated immediately, 4 or 9 h after injection. As shown in Fig. 1 A, BSA remained in the cytoplasm for at least 9 h after injection, while cyclins E and A1 translocated to the nucleus. Within 4 h, virtually all of the cyclin E and 50% of the cyclin A1 had translocated to the nucleus; the great majority of cyclin A1 was nuclear by 9 h. In contrast, cyclin B1 injected into the cytoplasm remained there for at least 18 h, consistent with its normally interphase cytoplasmic localization. A truncated form of cyclin B1 protein, which eliminates the NES (cycB1 121–397), entered the nucleus, albeit more slowly than cyclins E and A1; somewhat less than 50% of the injected protein had accumulated in the nucleus 9 h after injection. These data suggest that the nuclear import rates of all three cyclins are different, raising the possibility that they are recognized by the import machinery with different affinities or that distinct import pathways are used. Indeed, when we added fluorescein-conjugated, recombinant GST-cyclins B1 and E to Xenopus egg extracts containing ∼1,000 nuclei/μl, fluorescent cyclin E was clearly transported into nuclei more efficiently than cyclin B1, even after removal of the cyclin B1 NES (data not shown). We selected these cyclins, the fastest and slowest for nuclear import, for further study. Interestingly, these cyclins form complexes with different catalytic subunits. Moreover, in combination, Cdk2/cyclin E and Cdc2/cyclin B1 complexes are fully capable of driving multiple rounds of S phase and M phase in Xenopus egg extracts.

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