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• W1971747135 abstract "Article15 May 2000free access Cyclin E-mediated elimination of p27 requires its interaction with the nuclear pore-associated protein mNPAP60 Daniel Müller Daniel Müller Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Katja Thieke Katja Thieke Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Andrea Bürgin Andrea Bürgin Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Achim Dickmanns Achim Dickmanns Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Martin Eilers Corresponding Author Martin Eilers Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Daniel Müller Daniel Müller Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Katja Thieke Katja Thieke Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Andrea Bürgin Andrea Bürgin Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Achim Dickmanns Achim Dickmanns Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Martin Eilers Corresponding Author Martin Eilers Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany Search for more papers by this author Author Information Daniel Müller1, Katja Thieke1, Andrea Bürgin1, Achim Dickmanns2 and Martin Eilers 1 1Institute for Molecular Biology and Tumour Research, University of Marburg, Emil Mannkopff-Strasse 2, 35033 Marburg, Germany 2Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2168-2180https://doi.org/10.1093/emboj/19.10.2168 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Cdk2 inhibitor, p27Kip1, is degraded in a phosphorylation- and ubiquitylation-dependent manner at the G1–S transition of the cell cycle. Degradation of p27Kip1 requires import into the nucleus for phosphorylation by Cdk2. Phosphorylated p27Kip1 is thought to be subsequently re-exported and degraded in the cytosol. Using two-hybrid screens, we now show that p27Kip1 interacts with a nuclear pore-associated protein, mNPAP60, map the interaction to the 310 helix of p27 and identify a point mutant in p27Kip1 that is deficient for interaction (R90G). In vivo and in vitro, the loss-of-interaction mutant is poorly transported into the nucleus, while ubiquitylation of p27R90G occurs normally. In vivo, co-expression of cyclin E and Cdk2 rescues the import defect. However, mutant p27Kip1 accumulates in a phosphorylated form in the nucleus and is not efficiently degraded, arguing that at least one step in the degradation of phosphorylated p27Kip1 requires an interaction with the nuclear pore. Our results identify a novel component involved in p27Kip1 degradation and suggest that degradation of p27Kip1 is tightly linked to its intracellular transport. Introduction The p27Kip1 protein has a dual role in the G1 phase of the cell cycle: it inhibits Cdk2 complexes in resting cells and during the G1 phase of the cell cycle, and promotes assembly and nuclear import of cyclin D–Cdk4 complexes (for review, see Sherr and Roberts, 1999). At the end of the G1 phase, p27Kip1 is degraded. Degradation of p27Kip1 is sensitive to inhibitors of proteasome function, suggesting that degradation occurs via the ubiquitin pathway (Pagano et al., 1995). Ubiquitylated forms of p27Kip1 have been detected in vivo and degradation and ubiquitylation have been reconstituted in vitro using partially purified systems (Montagnoli et al., 1999; Nguyen et al., 1999; Shirane et al., 1999). Two mechanisms are known that ensure the correct timing of p27Kip1 degradation. First, p27Kip1 is a substrate of cyclin E–Cdk2 kinase and is phosphorylated by Cdk2 mainly, but not exclusively, at Thr187 (Müller et al., 1997; Sheaff et al., 1997; Vlach et al., 1997). In vivo, degradation of p27Kip1 requires active Cdk2 kinase and mutation of Thr187 inhibits degradation; in vitro, phosphorylation of Thr187 is required for ubiquitylation and subsequent degradation of p27Kip1 (Montagnoli et al., 1999; Tsvetkov et al., 1999). Together, the findings show that active cyclin–Cdk2 complexes during the G1 phase are required for elimination of p27Kip1. Secondly, skp2 has been identified as a candidate component of the E3 ligase complex that ubiquitylates p27Kip1 (Carrano et al., 1999). In vivo, skp2 function is required for p27Kip1 degradation (Carrano et al., 1999). Ectopic expression of skp2 in quiescent cells stimulates entry into S phase of the cell cycle and promotes degradation of p27Kip1, providing evidence that the amount of skp2 is rate limiting for degradation of p27Kip1 (Sutterlüty et al., 1999). In normal cells, expression of skp2 is restricted to the S phase (Zhang et al., 1995; Lisztwan et al., 1998). Although the mechanisms underlying this restriction are not known, the findings suggest that regulation of skp2 expression is a second mechanism that contributes to the correct timing of p27Kip1 degradation. In order to exert its function during G1 and to be degraded at the end of G1, p27Kip1 needs to be imported into the nucleus (Reynisdottir and Massague, 1997; Tomoda et al., 1999). Phosphorylation of p27Kip1 by Cdk2 is restricted to the cell nucleus due to the nuclear localization of CAK (Darbon et al., 1994), which may account for the requirement for nuclear import in degradation. A nuclear localization signal (NLS) has been assumed to be responsible for transport of p27Kip1 into the nucleus, since deletion of the C-terminus of p27Kip1 including the NLS abolishes import (Reynisdottir and Massague, 1997; Tomoda et al., 1999). Recently, a component of the signalosome, Jab1, has been found to interact with p27Kip1 and promote its degradation. Jab1 interacts with p27Kip1 and promotes its export from the nucleus, suggesting that degradation of phosphorylated p27Kip1 occurs in the cytosol (Tomoda et al., 1999). The signals mediating nuclear export of p27Kip1 are unknown. Using two-hybrid cloning we have identified an interaction between p27Kip1 and a nuclear pore-associated protein and provide evidence that this interaction is required for nuclear import of p27Kip1 and for degradation of phosphorylated p27Kip1 after nuclear import. Our findings identify a new component involved in p27Kip1 metabolism, support the notion that degradation of phosphorylated p27Kip1 requires export from the nucleus and have implications for the mechanisms by which intracellular transport of p27Kip1 is regulated. Results In order to identify novel proteins that interact with p27, we screened a mouse embryo cDNA library using full-length mouse p27 as bait. Of 500 positive clones, all except two coded for known cyclins (not shown); the remaining two were identical and encoded a novel p27-interacting protein (Figure 1A). The longest cDNAs that were isolated from a Balb/c-3T3 library encode a protein of 466 amino acids; the fragment recovered in the two-hybrid screen corresponds to amino acids 121–252 of the full-length protein. Western blotting using affinity-purified antibodies raised against this protein reveals an endogenous protein in a number of different cell lines; the endogenous protein comigrates with a protein expressed from a cDNA clone by transient transfection, demonstrating that a full-length cDNA was isolated (Figure 1B). Immunofluorescence experiments using these antibodies detected the endogenous protein in a speckled pattern at the nuclear periphery highly reminiscent of nuclear pore proteins or nuclear transport factors; mNPAP60 expressed by transient transfection showed a very similar distribution (Figure 1C). Database searches revealed that a rat cDNA homologous to the clones we had isolated had been described previously; the rat cDNA encodes a nucleoporin termed NPAP60 (Fan et al., 1997). We therefore refer to our clone as m(ouse)NPAP60. Both clones differ by a small sequence divergence in the N-terminus and by the presence of a putative RAN-binding domain in the C-terminus of the clone we have recovered, suggesting that the rat clone may possibly represent a shorter splice variant (see Supplementary figure 1, available at The EMBO Journal Online). Weak similarities to other nucleoporins were found, mainly within the putative RAN-binding domain. Figure 1.Identification of mNPAP60 as a p27-interacting protein. (A) Summary of yeast two-hybrid interaction data. Plasmids expressing the indicated chimeras were transformed into yeast strains and β-galactosidase activity was determined by a semi-quantitative filter assay. (B) Western blot documenting the expression of endogenous mNPAP60 in rodent fibroblast cell lines (left lanes). The right lanes contain extracts from HeLa cells transfected with vector alone or with an expression vector encoding mNPAP60. The antibody raised against mouse NPAP does not cross-react with human NPAP. (C) Immunofluorescence of cultured RAT1 cells using an affinity-purified antiserum raised against mNPAP60 (left). The right panel shows the localization of mNPAP60 expressed by transient transfection in HeLa cells. The middle panel shows a control after pre-incubation of the antiserum with a GST–mNPAP60 fusion protein. The pictures are a false-colour superimposition of the 4′,6-diamidino-2-phenylindole-stained nuclei (coloured in blue) and anti-mNPAP60 fluorescence (coloured in red). (D) In vitro binding of 35S-labelled p27 to GST–mNPAP60(121–252). The upper panel shows Coomassie-stained gels of the input into the in vitro binding reactions together with a molecular weight marker. BSA was added to reduce background binding. The middle panel shows a fluorography demonstrating specific binding of p27 to GST–mNPAP60. The lower panel shows reduced binding of p27 to GST–mNPAP60 after pre-incubation of p27 with cyclin E–Cdk2 complexes purified from baculovirus. The input lanes correspond to 10% of the total amount of p27 loaded on to the GST beads. (E) In vitro binding reactions from cell lysates. Detergent lysates from RAT1-MycER cells were prepared with or without heat treatment and incubated with GST–mNPAP60 as above. Western blots of the input lanes (10% of loading) and the recovered beads are shown. (F) Co-immunoprecipitation of p27 with anti-mNPAP60 antibodies. HeLa cells were transfected with the expression plasmids indicated, lysates prepared and immunoprecipitated with either antiserum against mNPAP60 or preimmune serum. Shown are Western blots probed with the antibodies indicated. N.S. reflects a non-specific band present in all immunoprecipitates. mNPAP60 migrates just above the IgG heavy chain on SDS gels; since the same antibody is used for both immunoprecipitation and Western blotting, the heavy chain is visible below the mNPAP60 bands in the immunoprecipitations. (G) Co-immunoprecipitation of endogenous mNPAP60 with anti-p27, but not with control antibodies from lysates of NIH 3T3 cells. The input represents 5% of cell extract used for the precipitation. Download figure Download PowerPoint A number of controls established that mNPAP60 interacts specifically with p27 in the yeast two-hybrid assay (Figure 1A). In order to verify that p27 and mNPAP60 interact outside the context of two-hybrid experiments, we generated and purified a glutathione S-transferase (GST)–mNPAP60 chimeric protein and incubated it with 35S-labelled p27 translated in a reticulocyte lysate (Figure 1D). In these experiments, p27 interacted efficiently with GST–mNPAP60, but not with equal amounts of GST alone. In similar experiments, no specific interaction of mNPAP60 was seen with cyclin D or E or with p21Waf1 (data not shown). p27 present in lysates of RAT1-MycER cells under conditions where it is bound to Cdk2 and Cdk4 (as judged by immunoprecipitation and gel filtration; not shown) did not interact with GST–mNPAP60. In contrast, p27 extracted from such lysates by a brief heat treatment specifically bound to GST–mNPAP60, suggesting that binding of p27 to Cdk2 and Cdk4 complexes prevents interaction with mNPAP60 in vitro (Figure 1E). Consistent with this notion, pre-incubation of p27 with cyclin E–Cdk2 complexes purified from insect cells infected with recombinant baculovirus abolished the interaction with GST–mNPAP60 (Figure 1D). An interaction between mNPAP60 and p27 was also detected by immunoprecipitation from cellular lysates (Figure 1F). HeLa cells were transfected with CMV-based expression vectors encoding p27 and mNPAP60 and detergent lysates were precipitated with either anti-mNPAP60 antibodies or preimmune antiserum. In these experiments, p27 was detected in anti-mNPAP60 immunoprecipitates when both proteins were co-expressed. No p27 was detected in precipitates using preimmune serum; also, p27 was not detected in anti-mNPAP60 immunoprecipitates from cells in which mNPAP60 had not been expressed. We noticed that expression of large amounts of mNPAP60 diminished expression of p27; addition of the proteasome inhibitor LLNL partly reversed this effect (not shown). Close inspection of the gels suggested that mNPAP60 associated preferentially with a slower- migrating, phosphorylated form of p27 (not shown). Therefore, we repeated the experiment with a mutant of p27 that lacks the Cdk2 phosphorylation site (T187V). In contrast to the two-hybrid situation (see below), this mutation diminished the association of p27 with mNPAP60 (Figure 1F). Potentially, therefore, proteins that specifically recognize the phosphorylated form of p27 stabilize the p27–mNPAP60 complex in vivo. The antibodies we have raised do not detect the human mNPAP60 protein. To determine whether endogenous mNPAP interacts with p27, lysates of NIH 3T3 cells were immunoprecipitated with anti-p27 and control antibodies. In the precipitates, mNPAP60 could be detected in anti-p27, but not in control precipitates, demonstrating that the endogenous proteins associate with each other (Figure 1G). To map the interaction surface between p27 and mNPAP60, progressive N- and C-terminal deletions of p27 were tested in the two-hybrid assay and by in vitro pull-down experiments (Figure 2A). Both assays gave consistent results, which map the N-terminal border of the interaction domain between amino acids 69 and 91 of p27 and the C-terminal border between amino acids 85 and 94. An internal deletion mutant of p27 spanning amino acids 85–98 failed to interact with mNPAP60 both in vitro and in two-hybrid assays (Figure 2A). Thus, the interaction surface centres on the 310 helix of p27, overlapping the cdk2-binding domain of the protein. This is consistent with the in vitro observations demonstrating that cdk2 and mNPAP compete for binding to p27. Figure 2.Isolation of a mNPAP60 loss-of-interaction mutant of p27. (A) Summary of two-hybrid data (left) and in vitro interaction assays (right) documenting the interaction of C-terminal, N-terminal and internal deletion mutants of p27 with mNPAP60. The assays were carried out as described in the legend to Figure 1. p27Δ91–197 lacks three of five methionines and is, therefore, labelled more weakly in these experiments. The right panel also documents the reduced interaction of the p27R90G mutant with GST–mNPAP60 in vitro. (B) Western blot documenting co-immunoprecipitation of p27wt, but not of p27R90G after co-expression with mNPAP60 in HeLa cells. Assays were carried out as described in the legend to Figure 1. (C) Inhibition of cyclin E–Cdk2 kinase activity by increasing amounts of p27. His-tagged recombinant p27wt and p27R90G proteins were purified and incubated with cyclin E–Cdk2 complexes isolated from baculovirus lysates. Histone H1 was used as a substrate. Triplicate assays are shown and were used to calculate the Ki values shown on the right. (D) The proteins indicated were purified and equal amounts subjected to phosphorylation by immunoprecipitated cyclin E–Cdk2 kinase. Shown is an autoradiogram of triplicate assays. The control designated ‘-cE/Cdk2’ contains p27wt protein and a mock precipitate using no antibody. Download figure Download PowerPoint In order to identify a mutant of p27 that specifically disrupts interaction with mNPAP60, a loss-of-interaction screen was performed in yeast. p27 was mutagenized using mismatch PCR in the presence of manganese ions and transformed in Escherichia coli. A total of 950 colonies were picked and the plasmids isolated were tested in a two-hybrid reaction with mNPAP60; 179 candidate negative colonies were selected, the plasmid isolated and re-transformed. From these, 22 plasmids were isolated, which encoded p27 proteins that failed to interact with mNPAP60. The plasmids were retested in a two-hybrid assay using cyclin D1 as prey and the p27 open reading frames of these plasmids were sequenced. Three plasmids were recovered that encoded mutant p27 proteins that interacted with cyclin D1 but failed to interact with mNPAP60 (see Supplementary figure 2). All three carried a single point mutation at position 90 (R90G). The R90G mutation was not present in any of the other plasmids recovered. In this screen, the mutations we recovered were exclusively A to G transitions (not shown). Therefore, the specificity of the arginine (AGG) to glycine (GGG) change is almost certainly due to the mutagenesis protocol we used. In wild-type p27, the arginine residue that is affected by the mutation forms a salt bridge with a glutamic acid residue at position 85; thus, it is very likely that this mutation at position 90 will affect the stability of the 310 helix (Russo et al., 1996). For all subsequent experiments, this mutation was re-introduced in a wild-type p27 plasmid and the plasmid was sequenced to ensure that it carries this single mutation. The localization of the mutation relative to other mapped interactions in the p27 sequence and within the p27 crystal structure is shown in Supplementary figure 2. To test whether the mutation indeed specifically disrupts the interaction with mNPAP60, we used in vitro pull-down experiments as before: in these experiments, p27R90G failed to interact with GST–mNPAP60 (Figure 2A). Also, p27R90G expressed by transient transfection in HeLa cells together with mNPAP60 failed to be precipitated by antibodies directed against mNPAP, in contrast to wild-type p27 (Figure 2B). We concluded that the R90G mutation disrupts association of p27 with mNPAP60 both in vitro and in vivo. To test whether the mutation affects interaction with cyclin E–Cdk2 complexes, p27 and p27R90G were expressed in E.coli as his-tagged recombinant proteins and incubated with cyclin E–Cdk2 complexes isolated from baculovirus-infected cells. In these assays, both proteins were indistinguishable. They inhibited cyclin E–Cdk2 kinase activity with identical Ki values and cyclin E–Cdk2 efficiently phosphorylated both proteins in vitro (Figure 2C and D). This result is in agreement with data obtained for other mutations in the 310 helix (Hashimoto et al., 1998). To investigate whether mNPAP60 is involved in the intracellular transport of p27, we expressed wild-type p27 and p27R90G by transient transfection in a number of cell lines and obtained similar results in HeLa, MCF7 and RAT1 cells. The results obtained in HeLa cells are summarized in Figure 3A. Examples of individual transfections are shown in Figure 3B. Using equal amounts of expression plasmid, a significant proportion of cells expressing p27R90G, but not p27wt, showed strong cytosolic staining in addition to nuclear staining. Similarly, the internal deletion mutant p27Δ85–98 mislocalized to the cytosol in a large fraction of the cells. Figure 3.Localization of p27 mutants in HeLa cells. (A) Quantitation of the results of several independent transfections into HeLa cells. (B) Examples of individual transfections. Download figure Download PowerPoint To determine the relevance of the NLS of p27 for nuclear transport in detail, the signal was disrupted by the replacement of three basic amino acids that constitute part of the NLS (R152A K153E R154A). This mutant showed a partial relocalization to the cytosol. A double mutant of p27 that carries both a mutation of the NLS and the R90G mutation mislocalizes to the cytosol in almost all of the transfected cells. The consistent mislocalization of both the R90G and the p27Δ85–98 mutations suggests a role for the mNPAP60–p27 interaction in the nuclear import of p27. To confirm this suggestion, recombinant p27wt and p27R90G proteins were fluorescently labelled and equal amounts of protein were injected into either the cytosol or the nucleus of HeLa cells (Figure 4A). When injected into the cytosol, wild-type p27 was rapidly imported into the cell nucleus; half-maximal import was observed 5 min after injection. In contrast, p27R90G was transported more slowly into the nucleus; in this assay the rate of import was ∼2-fold lower than that of wild-type p27. The data support the notion that the mNPAP60–p27 interaction has a functional role in nuclear import. When injected into the nucleus of HeLa cells, neither p27wt nor p27R90G were exported within the time frame of the experiment to a detectable degree; instead, the observable fluorescence decayed over the next 30 min. The data suggest that both proteins are retained in the nucleus. We could not, therefore, establish from these experiments whether mNPAP60 has a role in the nuclear export of p27 (see below). Figure 4.Characterization of the nuclear import of p27. (A) Recombinant p27wt and p27R90G proteins were fluorescently labelled and injected into the cytosol of exponentially growing HeLa cells. The time course of import is shown; between 50 and 150 cells were injected and evaluated for each time point. (B) Example of in vitro import reactions using fluorescently labelled p27 proteins and permeabilized HeLa cells. (C) Summary of results of in vitro import reactions. Download figure Download PowerPoint To characterize the mechanism of import in more detail, we tested both p27wt and p27R90G using in vitro nuclear import assays. An example of the results is shown in Figure 4B, and the results are summarized in Figure 4C. Wild-type p27 was imported efficiently into the nucleus in these assays. Import was strictly dependent on temperature and was abolished at 0°C; import was also abolished by addition of either wheat-germ agglutinin or a dominant-negative fragment of importin-β known to disrupt nucleoporin function (Kutay et al., 1997). Thus, nuclear import of p27 relies on a functional interaction with the nuclear pore and does not occur by free diffusion. This is expected, as recombinant p27 forms a dimer with an apparent molecular weight of 70 kDa in gel filtration assays (D.Müller and M.Eilers, unpublished data). Depletion of ATP, depletion of cytosol, addition of the importin-β-binding domain of importin-α (Klebe et al., 1995), addition of non-hydrolysable analogues of GTP or addition of a dominant-negative mutant of Ran (Weis et al., 1996) did not inhibit import. In parallel experiments, these treatments inhibited import of an NLS–bovine serum albumin (BSA) conjugate, as expected (not shown). Thus, nuclear import of p27 does not depend on the recognition of the NLS by importin-α or an energy-dependent mechanism. Import of p27R90G was strongly diminished relative to wild-type p27 under all conditions tested. We concluded that the in vitro nuclear import of p27 occurs via a process of facilitated diffusion across the pore and that interaction of p27 with mNPAP60 is required for this process. Upon expression from retroviral vectors, p27 inhibits proliferation of RAT1 cells (Vlach et al., 1996; Müller et al., 1997). To determine whether the interaction with mNPAP60 affects this function of p27 in vivo, we constructed retroviral vectors that express either wild-type or mutant p27 together with a puromycin resistance gene. RAT1 cells were infected with retroviral supernatants and pools of resistant cells selected; these pools were plated at low density to determine the rate of proliferation of these cells. Surprisingly, p27R90G consistently affected the rate of proliferation more strongly than wild-type p27 in repeated experiments (Figure 5A and B). Figure 5.Inhibition of cell proliferation by p27wt and p27R90G. RAT1 cells were infected with recombinant retroviruses expressing either p27wt or p27R90G. (A) The rate of cell proliferation of resistant cell pools relative to a pool of cells infected with a control (pbabe-puro) virus. (B) Average doubling time calculated from logarithmic plots of two independent infections. (C) Western blots documenting the expression of the proteins indicated and the amounts of cyclin A/p27 and cyclin E/p27 in exponentially growing cells infected with the viruses indicated. Download figure Download PowerPoint To explain this finding, we considered the possibility that ectopic expression of p27R90G might indirectly cause mislocalization of cyclins that lack an NLS such as cyclin A, but immunofluorescence experiments showed that this was not the case (not shown). However, in exponentially growing cells expressing p27R90G, levels of p27 were elevated relative to p27wt and elevated levels of p27R90G bound to cyclin E compared with either uninfected cells or cells infected with a virus expressing p27wt, indicating a defect in cyclin E-mediated elimination of p27 (Figure 5C). To confirm this suggestion, we co-expressed cyclin E and Cdk2 with different forms of p27 by transient transfection of HeLa cells (Figure 6A and B). In these assays, expression of cyclin E and Cdk2 induced efficient elimination of wild-type p27. This effect on p27 was fully reversible by addition of LLNL, an inhibitor of proteasomal degradation (not shown). In contrast, p27R90G was significantly more stable than wild-type p27; indeed, mutation at R90 stabilized p27 to a similar degree to a mutation of the major Cdk2 phosphorylation site, Thr187. Combining both mutations showed a strong synergy and yielded a mutant of p27 that is resistant to cyclin E-mediated elimination (Figure 6B). Figure 6.Cyclin E–Cdk2-induced degradation requires interaction of p27 with mNPAP60. HeLa cells were transfected with the indicated expression plasmids and analysed by Western blotting. (A) Control experiment documenting expression of cyclin E, Cdk2 and p27wt after transfection of the indicated amounts of plasmid; 5 μg of CMV–cyclin E and CMV–Cdk2 were used. (B) Repeat of the experiment using the indicated alleles of p27. Double lines indicate the presence of a phosphorylated form of p27; single arrows indicate absence of phosphorylated p27. (C) Cycloheximide-chase experiment documenting enhanced stability of phosphorylated p27R90G relative to p27wt. Transfections were carried out as in (B). The lower panels show a quantitation of the results and the upper panels show Western blots from a representative experiment. (D) p27R90G accumulates in the nucleus upon expression of cyclin E and Cdk2. Shown are immunofluorescence pictures of HeLa cells transfected with the expression vectors indicated together with a quantitation of the experiment. Download figure Download PowerPoint To show that the enhanced levels of p27R90G observed after expression of cyclin E–Cdk2 were due to a decreased degradation of the protein, cycloheximide-chase experiments were performed. Cycloheximide was added to a number of dishes 48 h after transfection, cells were harvested after different time points and the amount of remaining p27 was determined by Western blotting (Figure 6C). In these experiments, p27R90G was significantly more stable than p27wt. In particular, mutation at R90 stabilized the phosphorylated form of p27 relative to p27wt; in contrast, the mutation had little effect on the stability of non-phosphorylated p27, which is also degraded in these assays (albeit at a slower rate). From several such experiments, we calculated t1/2s of 60 min and of 240 min for the phosphorylated form of p27wt and p27R90G, respectively, the double mutant (T187VR90G) was degraded with a t1/2 of 250 min (not shown). Taken together, the data show that mutation of R90 inhibits degradation of phosphorylated p27. Since the p27R90G that accumulated upon co-expression of cyclin E and Cdk2 was phosphorylated (see arrows) and since cyclin E–Cdk2 complexes are only active in the cell nucleus (due to nuclear localization of CAK), the findings show that under these conditions interaction with mNPAP60 is required at a step after nuclear import. Indeed, the remaining fraction of p27R90G accumulated in the nucleus when co-expressed with cyclin E–Cdk2 (Figure 6D). We suggest that the NLSs present in cyclin E mediate efficient nuclear import of a ternary cyclin E–Cdk2–p27R90G complex in these assays. The failure of p27R90G to be correctly degraded might be a conseq" @default.
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• W1971747135 title "Cyclin E-mediated elimination of p27 requires its interaction with the nuclear pore-associated protein mNPAP60" @default.
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