Moore JD et al. (1999), Nuclear import of Cdk/cyclin complexes: identif...

XB-ART-13598

J Cell Biol 1999 Jan 25;1442:213-24.

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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 .

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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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Species referenced: Xenopus
Genes referenced: ccnb1 cdk1 cdk2 hnrnpa1 npm1 pold1 tbx2

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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.
	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.
	Figure 4. Interaction of Xenopus cyclins with importins. (A) Diluted interphase egg cytosol (final protein concentration of 10 mg/ml) was supplemented with 1/50 vol of reticulocyte lysates programmed to produce [35S]methionine-labeled variants of each of the following: Xenopus cyclin E, Xenopus cyclin B1, the truncated Xenopus cyclin B1 (121–397) mutant, or firefly luciferase. For controls, we used the known importin-α/β interactor nucleoplasmin (biotinylated for subsequent detection and added to extracts at 20 μg/ml), a similarly prepared “core” nucleoplasmin lacking an NLS (NPcore), or the core nucleoplasmin linked to the IBB domain of importin-α (NPcore-IBB). After addition of an ATP-regenerating system, extracts were incubated in batches with the GST-importin-β or GST resins. The beads were then pelleted, washed in buffer, and bound radiolabeled proteins were resolved by SDS-PAGE for detection by autoradiography or, in the case of the nucleoplasmins, for blotting with HRP-streptavidin. As egg extracts are rich in the adaptor subunit importin-α, immobilized GST-importin-β should be capable of interacting with basic NLS containing proteins such as nucleoplasmin. 1/10 the amount of supplemented extract used in the binding assay was loaded onto the gel as a control. (B) Coomassie-stained gel showing purity of human His-tagged cyclin E and cyclin B1, plus the same proteins copurified with their Cdc2 and Cdk2 catalytic subunit partners. Each lane contains 10 μg of recombinant protein. (C) The interaction between cyclin B1 and importin-β is direct and is not stimulated by Cdc2. His-tagged human cyclin B1 was purified on Ni2+NTA-agarose beads from overexpressing Sf9 cell lysates. For the purification of Cdc2/cyclin B1 complexes, the cyclin on the Ni2+ beads was incubated in Sf9 cell lysates overexpressing human HA-tagged Cdc2. After extensive washing, the immobilized cyclin B1 or Cdc2/ cyclin B1 complex was eluted with imidazole (250 mM) then concentrated in Centricon-30 microconcentrators and transferred to the PBS/cas-amino acids/Tween buffer (PAT buffer). The cyclin B1 or Cdc2/cyclin B1 complexes (15–20 μg/ml) were incubated with immobilized GST or GST-importin-β (∼1 μg protein/μl of beads). The beads were recovered after extensive washing; the bound proteins were then eluted and separated by SDS-PAGE (along with one-eighth of the relative amount of cyclin or Cdc2/cyclin complexes that had not been incubated with beads) and transferred to PVDF membranes. Cyclin B1 was detected by a murine mAb; Cdc2 was detected with sera raised against the conserved PSTAIRE epitope shared by Cdk2 and Cdc2. Blots probed with anti-PSTAIRE antiserum reveal that the cyclin B1 preparation was contaminated with very small amounts of Cdc2, presumably the native protein from the Sf9 cells. However, we estimate that our Cdc2/cyclin B1 preparation contains 25 times more Cdc2 than the cyclin B1 preparation. (D) Importin-α is required for efficient binding of cyclin E to importin-β. His-tagged human cyclin E (15 μg/ml in 180 μl PAT buffer) was incubated at 4°C with 20 μl of glutathione-Sepharose beads coupled to either GST or GST-importin-β, in the presence or absence of 100 μg/ml importin-α. The beads were recovered by centrifugation, washed extensively, and the bound proteins eluted and separated by SDS-PAGE. 1/10 the relative amount of cyclin E and recombinant importin-α included in the binding assays was also run on the gel as a control. Western blots were probed with antibodies against human cyclin E and Xenopus importin-α. (E) Both Cdc2 and Cdk2 require a cyclin partner to bind to importin-β. Extracts were made from Sf9 cells infected with recombinant baculoviruses inducing the overproduction of either human Cdc2 or Cdk2. The Sf9 lysates containing the Cdks were supplemented with 70 μg/ml importin-α and 10 μg/ml affinity-purified His-tagged cyclin B1 (for Cdc2) or E (for Cdk2), then incubated with immobilized GST or GST-importin-β. The human Cdks were detected with a mAb directed against their epitope tags (influenza hemagglutinin). As a control 1/10 of the relative amount of extracts before incubation with beads was loaded onto the gels along with the material eluted from the beads.
	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.
	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.
	Figure 7. Recombinant importins direct cyclin transport into nuclei from permeabilized HeLa cells. Isolated HeLa cell nuclei from digitonin-permeabilized cells were incubated in the presence of recombinant Ran and NTF2 proteins along with one of the following fluorescently labeled substrate proteins: GST-cyclin B1 (121–397), GST-cyclin E, GST-SV-40 T antigen NLS, or GST-M9. Assays were supplemented with recombinant importin-α, importin-β, or both or with MBP-transportin. In control assays, reticulocyte lysate was used as a source of all soluble transport factors. This was chosen rather than egg cytosol because it had been demonstrated previously to support robust import of transportin cargo in this assay.
	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.

References [+] :

Adam, Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. 1990, Pubmed, Xenbase