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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) A truncated cyclin E protein (GST-cyclin E 1–338)  does not bind Cdk2. Alignment of cyclin B1 with cyclin E, which  contains a longer extension beyond the second cyclin fold, revealed that a mutant of cyclin E defective in Cdk2 binding [analogous to the truncated cyclin B1 (121–373) mutant which does  not bind to Cdc2] might be made by removal of the COOH-terminal 71 amino acids of cyclin E. Here GST, GST-cyclin E, or GST-cyclin (1–338) proteins immobilized on glutathione-Sepharose  beads (20 μl) were incubated with 200 μl of interphase egg cytosol (diluted with PAT buffer to 10 mg/ml final protein concentration). Bead-bound proteins were recovered after pelleting and  extensive washing of the beads, eluted, and separated by SDS-PAGE. Western blots were probed with antibodies against Xenopus cyclin E or Cdk2. Untreated egg cytosol (1/10 the relative  amount used in the binding assays) was also run on the gel to  show the loading of Cdk2. Note that endogenous cyclin E from  the egg cytosol is not visible on the portion of the gel shown because it is considerably smaller than the cyclin E fusion proteins.  (B) COOH-terminal truncated versions of cyclin E and cyclin B1  that are unable to bind to their Cdk partners are imported into  nuclei of digitonin-permeabilized cells. Xenopus egg cytosol was  diluted 1:10 (4 mg protein/ml final concentration) to provide a  source of soluble transport factors. Fluorescein-labeled GST- cyclin E (1–338) and GST-cyclin B1 (121–373) were added to permeabilized cell assays at final concentrations of 50–100 μg/ml. Control reactions were performed on ice. These samples were processed  together with the samples shown in Fig. 1 B, which provide the  wild-type cyclin E and B1 controls.
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Figure 5: (A) A truncated cyclin E protein (GST-cyclin E 1–338) does not bind Cdk2. Alignment of cyclin B1 with cyclin E, which contains a longer extension beyond the second cyclin fold, revealed that a mutant of cyclin E defective in Cdk2 binding [analogous to the truncated cyclin B1 (121–373) mutant which does not bind to Cdc2] might be made by removal of the COOH-terminal 71 amino acids of cyclin E. Here GST, GST-cyclin E, or GST-cyclin (1–338) proteins immobilized on glutathione-Sepharose beads (20 μl) were incubated with 200 μl of interphase egg cytosol (diluted with PAT buffer to 10 mg/ml final protein concentration). Bead-bound proteins were recovered after pelleting and extensive washing of the beads, eluted, and separated by SDS-PAGE. Western blots were probed with antibodies against Xenopus cyclin E or Cdk2. Untreated egg cytosol (1/10 the relative amount used in the binding assays) was also run on the gel to show the loading of Cdk2. Note that endogenous cyclin E from the egg cytosol is not visible on the portion of the gel shown because it is considerably smaller than the cyclin E fusion proteins. (B) COOH-terminal truncated versions of cyclin E and cyclin B1 that are unable to bind to their Cdk partners are imported into nuclei of digitonin-permeabilized cells. Xenopus egg cytosol was diluted 1:10 (4 mg protein/ml final concentration) to provide a source of soluble transport factors. Fluorescein-labeled GST- cyclin E (1–338) and GST-cyclin B1 (121–373) were added to permeabilized cell assays at final concentrations of 50–100 μg/ml. Control reactions were performed on ice. These samples were processed together with the samples shown in Fig. 1 B, which provide the wild-type cyclin E and B1 controls.

Mentions: The experiments above suggested that nuclear import of Cdk/cyclin complexes might be promoted by direct interaction of the cyclin subunits with importins. However, previously published studies have indicated that the ability of mutant cyclins A and B3 to be imported into nuclei correlated with their ability to interact with Cdks (Maridor et al., 1993; Gallant and Nigg, 1994). Thus, it remained possible that Cdks might be in some way required for effective cyclin nuclear import. To determine if this was the case, we produced a recombinant mutant of cyclin B1 previously reported to lack the ability to interact with Cdc2 (cyclin B1 121–373; Stewart et al., 1994) and an analogous mutant of cyclin E unable to bind Cdk2 (cyclin E 1–338; Fig. 5 A). Both of these cyclins bound to importins as efficiently as their wild-type counterparts (data not shown). Moreover, fluoresceinated variants of these proteins were imported efficiently into nuclei of digitonin-permeabilized cells (Fig. 5 B; for the purposes of comparison, note that the control cyclin B1 and cyclin E import assays from this experiment are shown with the same exposure time in Fig. 1 B). These data suggest that Cdk binding is entirely dispensable for cyclin nuclear import, in accordance with our observation that the major determinants of importin binding reside within the cyclin subunit of the cyclin/Cdk complex. As predicted by these data, we have found that depletion of all of the Cdk2 and >90% of the Cdc2 from egg cytosol using p13 beads had no deleterious effect on nuclear import of either cyclin E or cyclin B1 (data not shown).


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) A truncated cyclin E protein (GST-cyclin E 1–338)  does not bind Cdk2. Alignment of cyclin B1 with cyclin E, which  contains a longer extension beyond the second cyclin fold, revealed that a mutant of cyclin E defective in Cdk2 binding [analogous to the truncated cyclin B1 (121–373) mutant which does  not bind to Cdc2] might be made by removal of the COOH-terminal 71 amino acids of cyclin E. Here GST, GST-cyclin E, or GST-cyclin (1–338) proteins immobilized on glutathione-Sepharose  beads (20 μl) were incubated with 200 μl of interphase egg cytosol (diluted with PAT buffer to 10 mg/ml final protein concentration). Bead-bound proteins were recovered after pelleting and  extensive washing of the beads, eluted, and separated by SDS-PAGE. Western blots were probed with antibodies against Xenopus cyclin E or Cdk2. Untreated egg cytosol (1/10 the relative  amount used in the binding assays) was also run on the gel to  show the loading of Cdk2. Note that endogenous cyclin E from  the egg cytosol is not visible on the portion of the gel shown because it is considerably smaller than the cyclin E fusion proteins.  (B) COOH-terminal truncated versions of cyclin E and cyclin B1  that are unable to bind to their Cdk partners are imported into  nuclei of digitonin-permeabilized cells. Xenopus egg cytosol was  diluted 1:10 (4 mg protein/ml final concentration) to provide a  source of soluble transport factors. Fluorescein-labeled GST- cyclin E (1–338) and GST-cyclin B1 (121–373) were added to permeabilized cell assays at final concentrations of 50–100 μg/ml. Control reactions were performed on ice. These samples were processed  together with the samples shown in Fig. 1 B, which provide the  wild-type cyclin E and B1 controls.
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Related In: Results  -  Collection

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Figure 5: (A) A truncated cyclin E protein (GST-cyclin E 1–338) does not bind Cdk2. Alignment of cyclin B1 with cyclin E, which contains a longer extension beyond the second cyclin fold, revealed that a mutant of cyclin E defective in Cdk2 binding [analogous to the truncated cyclin B1 (121–373) mutant which does not bind to Cdc2] might be made by removal of the COOH-terminal 71 amino acids of cyclin E. Here GST, GST-cyclin E, or GST-cyclin (1–338) proteins immobilized on glutathione-Sepharose beads (20 μl) were incubated with 200 μl of interphase egg cytosol (diluted with PAT buffer to 10 mg/ml final protein concentration). Bead-bound proteins were recovered after pelleting and extensive washing of the beads, eluted, and separated by SDS-PAGE. Western blots were probed with antibodies against Xenopus cyclin E or Cdk2. Untreated egg cytosol (1/10 the relative amount used in the binding assays) was also run on the gel to show the loading of Cdk2. Note that endogenous cyclin E from the egg cytosol is not visible on the portion of the gel shown because it is considerably smaller than the cyclin E fusion proteins. (B) COOH-terminal truncated versions of cyclin E and cyclin B1 that are unable to bind to their Cdk partners are imported into nuclei of digitonin-permeabilized cells. Xenopus egg cytosol was diluted 1:10 (4 mg protein/ml final concentration) to provide a source of soluble transport factors. Fluorescein-labeled GST- cyclin E (1–338) and GST-cyclin B1 (121–373) were added to permeabilized cell assays at final concentrations of 50–100 μg/ml. Control reactions were performed on ice. These samples were processed together with the samples shown in Fig. 1 B, which provide the wild-type cyclin E and B1 controls.
Mentions: The experiments above suggested that nuclear import of Cdk/cyclin complexes might be promoted by direct interaction of the cyclin subunits with importins. However, previously published studies have indicated that the ability of mutant cyclins A and B3 to be imported into nuclei correlated with their ability to interact with Cdks (Maridor et al., 1993; Gallant and Nigg, 1994). Thus, it remained possible that Cdks might be in some way required for effective cyclin nuclear import. To determine if this was the case, we produced a recombinant mutant of cyclin B1 previously reported to lack the ability to interact with Cdc2 (cyclin B1 121–373; Stewart et al., 1994) and an analogous mutant of cyclin E unable to bind Cdk2 (cyclin E 1–338; Fig. 5 A). Both of these cyclins bound to importins as efficiently as their wild-type counterparts (data not shown). Moreover, fluoresceinated variants of these proteins were imported efficiently into nuclei of digitonin-permeabilized cells (Fig. 5 B; for the purposes of comparison, note that the control cyclin B1 and cyclin E import assays from this experiment are shown with the same exposure time in Fig. 1 B). These data suggest that Cdk binding is entirely dispensable for cyclin nuclear import, in accordance with our observation that the major determinants of importin binding reside within the cyclin subunit of the cyclin/Cdk complex. As predicted by these data, we have found that depletion of all of the Cdk2 and >90% of the Cdc2 from egg cytosol using p13 beads had no deleterious effect on nuclear import of either cyclin E or cyclin B1 (data not shown).

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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