Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.
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.
Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
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.
- CDC2 Protein Kinase/genetics/metabolism*
- CDC2-CDC28 Kinases*
- Cyclin B/genetics/metabolism*
- Cyclin E/genetics/metabolism*
- Cyclin-Dependent Kinases/genetics/metabolism*
- Protein-Serine-Threonine Kinases/genetics/metabolism*
- Binding Sites
- Biological Transport
- Cell Nucleus/metabolism
- Cyclin B1
- Cyclin-Dependent Kinase 2
- Nuclear Proteins/metabolism
- Recombinant Fusion Proteins/genetics/metabolism
- Xenopus Proteins
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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.
Since the above data indicated that cyclin E nuclear import required importin-α, we wished to determine whether cyclin E could interact physically with the importin-α/β heterodimer used to import classical NLS-containing proteins. For this purpose, resins linked to GST or GST- importin-β were incubated in interphase Xenopus egg cytosol supplemented with in vitro translated, radiolabeled cyclins, pelleted, washed, and resolved by autoradiography. As shown in Fig. 4 A, cyclin E behaved like the NLS-containing control protein, nucleoplasmin (biotinylated for detection with HRP-streptavidin), binding specifically to importin-β. Similar interactions were observed using a nucleoplasmin variant in which the NLS had been removed and replaced with the IBB domain of importin-α (NPcore-IBB), whereas the NPcore alone, or luciferase, both lacking nuclear localization signals, did not bind any transport factors. The interaction of nucleoplasmin with GST-importin-β beads indicates that importin-α binding proteins are capable of interacting (presumably indirectly via importin-α provided by the extract) with immobilized importin-β in this system. Indeed, given the requirement for importin-α in cyclin E nuclear import (Fig. 3 B), we suspected that importin-α from the extract mediated cyclin E–importin-β interactions (a point we will return to below). Since importin-α did not appear to be required for cyclin B1 nuclear import, we were surprised to find that the importin-β resin was also able to retrieve radiolabeled cyclin B1 from egg extracts (Fig. 4 A). These data suggested that cyclin B1 might bind directly to importin-β.