HA95 and LAP2β coprecipitate in interphase
HA95 coprecipitates with a protein complex containing LAP2β from the transformed human B cell line Bjab in interphase (Martins et al., 2000
). We now show that in Bjab cells, HA95 and LAP2β coprecipitated in interphase but not at mitosis, regardless of the precipitating antibody ( A). Immunoblots of the nonprecipitated (S) fractions indicate that in interphase, HA95 coprecipitated all detectable LAP2β, whereas LAP2β did not precipitate all HA95 ( A). Immunoprecipitation of an HA95–Myc fusion protein stably expressed in Bjab cells using an anti-Myc antibody also coprecipitated LAP2β in interphase but not at mitosis ( B). These results suggest a cell cycle–dependent interaction of HA95 with LAP2β; however, not all HA95 resides in a complex with LAP2β.
Figure 1. HA95 coprecipitates with LAP2β in interphase. (A) HA95 or LAP2β was immunoprecipitated (IP) from interphase and mitotic Bjab cells. Precipitates (P) and supernatants (S) were immunoblotted using anti-LAP2β or anti-HA95 antibodies. (more ...)
The strength of interaction between HA95 and LAP2β within the complex was evaluated by a 30-min extraction of anti-HA95 immune precipitates (HA95-IPs) with 0–1.25 M NaCl. C shows that the interaction resisted a 0.75 M NaCl wash but was completely disrupted with 1.25 M NaCl. High salt resistance of the interaction suggests a strong association between HA95 and LAP2β, confirming our earlier cross-linking data (Martins et al., 2000
Mapping of the HA95-binding domains of LAP2β
To map the domains of LAP2β involved in the interaction with HA95, GST–LAP2β fusion polypeptides were produced ( A) (Furukawa et al., 1995
). Binding of each peptide to HA95–Myc was determined in GST precipitations after incubation of the peptides in a nuclear extract from Bjab cells expressing HA95–Myc. Control extracts were incubated with GST or glutathione beads alone. GST precipitates were immunoblotted using anti-Myc antibodies. B shows that LAP2β(1–452), (1–397), (1–298), (137–373), (137–298), (243–397), (243–373), (299–397), and (299–373) precipitated HA95–Myc. In contrast, LAP2β(1–193), (1–85), or (194–298) did not precipitate HA95–Myc. Thus, a first HA95-binding domain localizes to amino acids 137–242 of LAP2β and a second domain coincides with the lamin B–binding domain at residues 299–373. We designated these domains HA95-NBD (for HA95 NH2
-terminal binding domain) and HA95-CBD (HA95 COOH-terminal binding domain), respectively.
Figure 2. LAP2β interacts with HA95 via two distinct domains. (A) GST–LAP2β deletion peptides. (B) Indicated peptides were incubated in nuclear extracts of Bjab cells expressing HA95–Myc and sedimented by GST precipitation. Binding (more ...)
The Myc tag itself did not bind GST or LAP2β. LAP2β(137–373), which binds HA95–Myc, did not coprecipitate a stably expressed Myc-tagged p70S6 kinase ( C). Furthermore, anti-Myc antibodies coprecipitated LAP2β (1–397) but not the nonbinding fragment LAP2β(1–193) from a nuclear extract of Bjab cells expressing HA95–Myc (unpublished data). Altogether, the results indicate that HA95 interacts with recombinant LAP2β.
Direct association of LA2β with HA95 was demonstrated in an overlay assay. HA95-IP proteins were resolved by SDS-PAGE, blotted, and overlaid with 50 μM of each GST–LAP2β fragment. The same GST–LAP2β peptides found to precipitate HA95 also bound HA95 in the overlay, as detected with anti-GST antibodies ( D, top). Moreover, all LAP2β fragments containing the lamin B–binding domain (residues 299–373) bound to immunoprecipitated and immobilized lamin B ( D, bottom). However, two of the HA95-binding peptides, LAP2β(1–298) and LAP2β(137–298), did not bind lamin B, confirming the existence of an HA95-binding domain (HA95-NBD) distinct from the lamin B–binding region.
To determine whether HA95-binding LAP2β peptides would disrupt endogenous HA95–LAP2β association, dissociation experiments were performed using GST–LAP2β peptides harboring both HA95-binding domains (LAP2β [137–373]) or either HA95-NBD (LAP2β[137–298]) or HA95-CBD (LAP2β[299–373]). HA95-IPs were incubated for 1 h with 100 μM GST–LAP2β peptides, HA95-IPs were sedimented, and dissociation of LAP2β from HA95 was monitored by immunoblotting. LAP2β(137–373) completely dissociated LAP2β from HA95-IP ( E, lanes 1 and 2), however peptides containing HA95-NBD or HA95-CBD were ineffective (lanes 3–6). Nevertheless, two sequential 30-min incubations of HA95-IPs with LAP2β(137–298) followed by LAP2β(299–373) led to dissociation of the complex (lanes 7 and 8), and reversing the order of peptide addition produced similar results (lanes 9 and 10). We concluded that disruption of both HA95-binding domains was required for dissociation of LAP2β from HA95 in vitro. The data also argue that LAP2β(137–298) and LAP2β(299–373) peptides are capable of dissociating HA95 from HA95-NBD and HA95-CBD, respectively.
Inhibition of LAP2β binding to HA95 does not affect nuclear reassembly in vitro
To determine whether interaction between HA95 and LAP2β was involved in NE assembly, we assessed the ability of HA95-binding GST–LAP2β peptides to compete with LAP2β for membrane targeting to chromosomes. Purified HeLa nuclei were disassembled in mitotic extract. The resulting condensed chromosomes contained HA95 and BAF, but no A- or B-type lamins, LAP2β, or lamin B receptor (LBR) ( A). After preincubation with 10 μM of each GST–LAP2β peptide, chromosomes were sedimented and peptide binding was examined by immunofluorescence using anti-GST antibodies. All peptides except GST–LAP2β(194–298) bound chromatin ( B), as anticipated from their ability to bind HA95 (), DNA, or chromatin (see Introduction). Chromosomes were resuspended in interphase extract ( C, Input chrom.) under conditions promoting nuclear assembly. Nuclear morphology was examined by phase contrast microscopy and membrane labeling with DiOC6 after 2 h ( C). Without peptide or with GST alone, >80% of chromatin masses supported nuclear reformation. In contrast, LAP2β fragments (1–452), (1–397), (137–373), (243–397), (299–397), (243–373), and (299–373) inhibited nuclear assembly. Each of these peptides contained the lamin B–binding domain/HA95-CBD. Peptides that do not bind HA95 (nor lamin B) (LAP2β[1–193], [1–85], and [194–298]) did not block membrane assembly, neither did LAP2β(1–298) or (137–298), which both contain HA95-NBD.
Figure 3. Disruption of HA95 interaction with LAP2β does not interfere with nuclear assembly in vitro. (A) Purified HeLa nuclei (Nuc) were disassembled in mitotic extract, and chromosomes (Chr) were immunoblotted using the indicated antibodies. (B) Indicated (more ...)
To address whether disruption of the of HA95–LAP2β interaction altered global nuclear organization, localization of LAP2β, A- and B-type lamins, and BAF was analyzed by immunofluorescence in nuclei reconstituted in the presence of either no peptide, a peptide not binding to HA95 (LAP2β[1–193]), or peptides containing both HA95-NBD and HA95-CBD (LAP2β[137–373]), HA95-NBD only (LAP2β[137–298]), or HA95-CBD only (LAP2β[299–373]). Anti-GST immunolabeling shows peptide distribution in the resulting nuclei or chromatin masses ( D). Distribution of LAP2β, lamins A/C and B, or BAF was not distinctively altered in nuclei reconstituted with HA95-binding or nonbinding LAP2β peptides ( D). LAP2β targeting to chromatin and nuclear assembly of A- and B-type lamins, or lack thereof, were verified by immunoblotting nuclei or chromatin purified from the extract ( E). The blots also showed that levels of chromatin-associated BAF were not significantly altered by GST–LAP2β fragments ( E). We concluded that LAP2β fragments containing the lamin B–binding domain and HA95-CBD prohibit nuclear reformation. However, peptides harboring HA95-NBD only are not inhibitory and do not notably affect the distribution of NE proteins or BAF in the reassembled nuclei. Thus, we could not attribute an effect of HA95 binding per se on NE assembly in vitro.
LAP2β(137–298) inhibits initiation of DNA replication in intact nuclei in vitro
To determine whether interphase nuclear functions were affected by the HA95–LAP2β association, we monitored the effect of disrupting the LAP2β–HA95 interaction on DNA replication in purified G1-phase nuclei. To this end, we adapted an in vitro replication assay from that of Krude et al. (1997)
Nuclei were isolated from G1-phase HeLa cells. GST–LAP2β peptides were introduced into the nuclei after mild treatment with lysolecithin. Lysolecithin was previously shown not to affect dynamic properties of isolated nuclei in in vitro nuclear disassembly assays (Collas et al., 1999
). Peptides were taken up by ~90% of the nuclei, as shown by immunofluorescence using anti-GST antibodies ( A; GST–LAP2β[1–452] is shown). Control and peptide-loaded nuclei were incubated for 3 h in a concentrated (25–30 mg/ml) nuclear and cytosolic extract from S-phase HeLa cells containing [α32
P]dCTP, dNTPs, GTP, and an ATP-regenerating system to promote replication. Under these conditions, G1 nuclei loaded with GST–LAP2β(1–452) were capable of importing an exogenous BSA–nuclear localization signal conjugate (unpublished data) or the replication factor Cdc6 ( B). Import was ATP and GTP dependent and blocked by preincubation of the nuclei with antibodies against nucleoporins ( B, mAb414). These results indicate that import took place through nuclear pores rather than passively through a damaged NE, and confirmed a previous report of physiological import of transcription factors by nuclei purified as previously described (Landsverk et al., 2002
). We also tested whether peptides containing HA95-NBD or HA95-CBD introduced into G1 nuclei would inhibit nuclear import under the conditions described above, as this would be expected to affect DNA replication. C shows that none of the peptides distinctly impaired import of Cdc6. Notably, import was permitted by the HA95-NBD–containing peptide LAP2β(137–298) and blocked by mAb414. Lastly, the nuclear DNA did not undergo any detectable degradation upon incubation of the G1 nuclei in the extract at 4°C or 37°C, as judged by TUNEL analysis ( C) and DNA agarose gel electrophoresis ( D). These results indicate that isolated G1 nuclei are functional in import, can be manipulated to introduce peptides, and do not undergo detectable DNA degradation in S-phase extract, and that LAP2β fragments containing either HA95-binding domain do not block nuclear import of Cdc6 in vitro.
Figure 4. Characterization of nuclei used in the in vitro DNA replication assay. (A) Nuclei purified from HeLa cells in G1 phase were loaded with GST–LAP2β(1–452). Peptide uptake was analyzed by immunofluorescence using anti-GST antibodies. (more ...)
Figure 5. LAP2β(137–298) inhibits initiation of DNA replication in G1 nuclei. (A) Peptide-loaded G1 nuclei were incubated in S-phase extract containing [α32P]dCTP, dNTPs, GTP, and an ATP-regenerating system. [α32P]dCTP incorporation (more ...)
Having characterized the nuclei, the effect of GST–LAP2β peptides on replication was examined. Replication was assayed by incorporation of [α32P]dCTP, DNA electrophoresis, and phosphorImaging ( A). Absence of GST–LAP2β peptide or loading GST alone into G1 nuclei did not hinder replication. As expected, DNA synthesis was blocked with 50 μM aphidicolin in the extract. However, LAP2β(1–452) and (1–397) abolished replication and peptides containing HA95-CBD (LAP2β[243–397], [299–397], [243–373], and [299–373]) impaired replication efficiency by ~50%. Furthermore, LAP2β(1–298), (1–193), (137–373), and (137–298) inhibited replication, whereas LAP2β(194–298) did not. We also ruled out the possibility that the DNA replication signal detected in G1 nuclei represented an elongation phase in already replicating nuclei, because G1 nuclei incubated in extract from G0-arrested cells did not replicate ( B).
Initiation of replication requires the cyclin A–Cdk2 complex (Stoeber et al., 1998
). Thus, to provide evidence that DNA synthesis in G1 nuclei was due to true initiation of replication, we showed that DNA synthesis was inhibited with 10 μM of the Cdk2 inhibitor, olomoucine, in the assay (Stoeber et al., 1998
) ( B). Similar results were obtained with 500 μg/ml dimethylaminopurine, a nonspecific protein kinase inhibitor (unpublished data). Altogether, the results indicate that peptides containing HA95-CBD partially inhibit replication in G1 nuclei. However, those containing HA95-NDB completely abolish replication. We ruled out an involvement of the GCL-binding domain of LAP2β (residues 219–298) in inhibition of replication by fragment 137–298 because LAP2β(194–298) was not inhibitory.
As described earlier in the nuclear reconstitution experiment, none of the peptides altered the immunofluorescence labeling pattern of LAP2β, A- and B-type lamins, or BAF in the G1 nuclei examined (unpublished data; see also below). In addition, Western blot analysis of the G1 nuclei loaded with the peptides shows that neither peptide affected BAF levels (or HA95) in the nuclei ( C). As LAP2β(1–193) and (137–298) block replication whereas LAP2β(194–298) and (1–85) allow replication ( D), the data suggest that region 137–193 of LAP2β is involved in replication initiation without affecting the distribution of NE proteins or the amount of BAF in the nuclei.
To establish that the LAP2β(137–298) peptide inhibited semiconservative DNA replication in G1 nuclei, BrdU substitution and density gradient centrifugation of 32P-labeled DNA was performed. G1 nuclei loaded with no peptide, GST alone, or LAP2β(137–298) were incubated for 3 h in S-phase extract containing BrdU and [α32P]dCTP to quantitate replication. DNA was analyzed by CsCl gradient centrifugation. In control nuclei, the cytosol promoted DNA synthesis, producing primarily hemisubstituted (heavy-light [HL]) DNA, indicative of semiconservative replication ( E). In contrast, no peak of hemisubstituted DNA was detected in nuclei loaded with LAP2β(137–298), consistent with replication inhibition detected by 32P incorporation. We concluded that LAP2β(137–298) interfered with semiconservative replication in G1 nuclei.
Disrupting LAP2β–HA95 interaction does not affect the elongation phase of DNA replication in vitro
We next determined whether the elongation phase of replication was also affected by LAP2β peptides. Nuclei isolated from S-phase HeLa cells were loaded with each GST–LAP2β peptide ( F, left; GST–LAP2β[1–452] is shown) and incubated in S-phase extract under conditions promoting replication. Remarkably, all peptides supported DNA synthesis to the same extent as a control without peptide ( F). In addition, to validate our DNA replication assay, we showed that DNA synthesis occurred in S-phase nuclei incubated in extract from G0 cells, reflecting the replicating state of these nuclei ( B). In contrast, G0 nuclei did not synthesize DNA in S-phase extract. Note, however, a faint 32P label in G0 nuclei in the S-phase extract, due to a minor proportion of slowly replicating nuclei in the G0 cell population (unpublished data).
HA95 coimmunoprecipitates with the Cdc6 protein in G1 phase
Initiation of DNA replication requires the assembly of preRCs at origins of replication in G1. The chromatin-bound ORC complex recruits Cdc6, which in turn promotes targeting of MCM proteins. HA95 and Cdc6 were found to coimmunoprecipitate from G1-phase HeLa cells; nevertheless, whereas anti-HA95 antibodies precipitated all detectable Cdc6, a substantial fraction of HA95 did not associate with the Cdc6 immune precipitate ( A). In S phase however, Cdc6 and HA95 did not coprecipitate ( A), despite the reported persistence of a fraction of Cdc6 on chromatin beyond G1 (Mendez and Stillman, 2000
Figure 6. Dissociation of HA95 from HA95-NBD in G1 elicits degradation of Cdc6. (A) HA95 (top three panels) or Cdc6 (bottom three panels) was immunoprecipitated from G1- or S-phase HeLa cells, and immune precipitates (P) and supernatants (S) were immunoblotted (more ...)
Disruption of the interaction between HA95 and LAP2β via HA95-NBD in G1 induces proteolysis of Cdc6
The role of Cdc6 on preRC assembly led us to investigate the fate of Cdc6 in G1 nuclei after disruption of the HA95–LAP2β association via HA95-NBD or HA95-CBD. GST–LAP2β peptides were introduced into purified G1 HeLa nuclei and nuclei were incubated in nuclear isolation buffer for 1 h. Immunoblotting analyses of the nuclei show that nuclei loaded with GST alone or with LAP2β(299–373) contained Cdc6 ( B, lanes 1–3). However, nuclei loaded with LAP2β(137–298) or LAP2β(1–452) harbored no detectable Cdc6 (lanes 4 and 5). Additionally, blots of the whole incubation mix (nuclei and buffer) revealed no Cdc6 either, indicating that Cdc6 was degraded under these conditions (lane 9; see below). Note that p53 was also degraded in nuclei containing peptides harboring HA95-NBD, however, Orc2, a component of the preRC, was not affected (lanes 4 and 5).
The ORC large subunit has recently been shown to be degraded by ubiquitin-mediated proteolysis in human cells (Mendez et al., 2002
). Thus degradation of Cdc6 by the 25S proteasome was examined. Inhibition of the proteasome by incubation of nuclei containing LAP2β(137–298) with 25 μM of the proteasome inhibitors LLnL or β-lactone during peptide loading and incubation in buffer prevented degradation of Cdc6 ( B, lanes 6 and 7). However, the calpain inhibitor LLM, used as a control, did not block Cdc6 proteolysis (lane 8). Inhibition of degradation of p53, a known substrate of ubiquitin-mediated proteolysis, with LLnL or β-lactone confirmed the efficacy of the inhibitors ( B). Introduction of LAP2β(137–298) in S-phase nuclei did not elicit degradation of Cdc6 (or p53 or Orc2; unpublished data). These results suggest that dissociation of HA95 from HA95-NBD in G1- but not S-phase nuclei triggers degradation of Cdc6 by the proteasome. Because p53 is also proteolyzed by LAP2β(137–298), the data also suggest that the peptide promotes degradation of a subset of nuclear proteins.
As G1 nuclei containing LAP2β(137–298) do not replicate DNA in S-phase extract, we determined whether proteasome inhibitors would rescue S-phase entry of these nuclei. G1 nuclei were loaded with LAP2β(137–298) in the presence of LLnL or β-lactone and incubated for 3 h in S-phase extract containing [α32P]dCTP under conditions promoting replication. C shows that LLnL and β-lactone relieved the inhibition of DNA replication imposed by LAP2β(137–298). The calpain inhibitor LLM, however, had no effect and, as expected, LLnL or β-lactone did not affect replication of LAP2β(299–373)-loaded G1 nuclei ( C). Therefore, inhibition of the proteasome enables entry of the G1 nuclei into a replication phase.
Injection of LAP2β(137–298) into G1 nuclei inhibits entry into S phase in vivo
The significance of HA95 interaction with HA95-NBD or HA95-CBD was further investigated in vivo by injections of ~5 nM GST–LAP2β peptides into the nuclei of HeLa cells in early G1 (2 h after release from mitotic arrest). Injections were verified by nuclear retention of a 150-kD FITC–dextran (see below and A).
Figure 8. Nuclear injection of GST–LAP2β(137–298) in G1 inhibits replication. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone together with a 150-kD FITC–dextran to (more ...)
We first assessed whether the distribution of INM and lamina proteins was altered in the injected nuclei. As seen earlier in in vitro–reconstituted nuclei, GST–LAP2β peptides were detected throughout the nucleus for the most part, with a propensity of the anti-GST antibody to decorate the nuclear periphery more strongly (, GST). This, however, was not specific for the peptide injected (, bottom three rows). Immunofluorescence analysis of peptide- and mock (buffer)-injected cells indicated that LAP2β and B-type lamins remained localized at the NE 2–3 h after injection with either peptide (). Similar results were obtained for LBR, emerin, and A-type lamins (unpublished data). Additionally, no alteration in the localization of BAF in peptide-injected and control cells was detected (). BAF remained distributed throughout the nucleoplasm with an enrichment around the periphery. Thus, we could not attribute a noticeable effect of intranuclear peptide injection in G1 on overall nuclear architecture.
Figure 7. Nuclear injection of GST–LAP2β(137–298) in G1 does not alter distribution of NE proteins or BAF. Nuclei of G1 HeLa cells were injected with 5 ng of the indicated GST–LAP2β peptide or GST alone, cultured for 2–3 (more ...)
To examine the effect of peptide injection in G1 on DNA replication, injected G1-phase cells were cultured with 10 μM BrdU for 10 h and DNA synthesis was monitored using anti-BrdU antibodies. (A and B) shows that 95% of mock-injected cells underwent DNA synthesis, which was inhibited by 50 μM aphidicolin. Likewise, cells injected with LAP2β(1–85) or GST alone replicated DNA. However, LAP2β(137–298) abolished DNA synthesis in 90% of the cells, whereas LAP2β(299–373) had no effect. Injection of LAP2β(137–298) in S-phase nuclei was not inhibitory (unpublished data), indicating that once DNA synthesis is initiated, disruption of the LAP2β–HA95 interaction via HA95-NBD has no effect.
Additional immunofluorescence analysis of injected G1 HeLa cells indicated that Cdc6 was undetectable in LAP2β (137–298)-injected cells, whereas intranuclear labeling was evident in noninjected cells ( C, arrow) or in cells injected with GST alone or LAP2β(299–373) ( C). Note that Orc2 was not degraded in LAP2β(137–298)-injected cells, as shown by intranuclear immunolabeling ( C). The results indicate that, as shown biochemically in vitro, LAP2β(137–298) inhibits S-phase entry in vivo. Inhibition correlates with the degradation of Cdc6, but is not due to a displacement of NE proteins or a change in BAF distribution during G1.