Correspondence to: Philippe Collas, Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, P.O. Box 1112 Blindern, 0317 Oslo, Norway. Tel:47-22-85-10-13 Fax:47-22-85-14-97 E-mail:philippe.collas{at}basalmed.uio.no.
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Abstract |
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Protein kinase A (PKA) and the nuclear A-kinaseanchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RII regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKAsignaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.
Key Words: AKAP, cAMP, chromosome condensation, mitosis, PKA
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Introduction |
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PROPER packaging of DNA into chromosomes is an essential process in preparation for mitosis. Chromosome condensation at mitosis requires DNA topoisomerase II (
cAMP-dependent protein kinase A (PKA) has been proposed to be a negative regulator of mitosis. PKA activity oscillates in cycling Xenopus egg extracts (
Biological effects of cAMP are mainly mediated by PKA types I and II in eukaryotic cells. The PKA type II holoenzyme complex consists of two catalytic (C) and two regulatory (RII or RIIß) subunits, which modulate the catalytic activity of PKA by binding and inactivating C (
The specificity of cellular and nuclear responses to cAMP is mediated by targeting of the RII subunit of PKA to discrete subcellular loci through associations with A-kinaseanchoring proteins or AKAPs ( has been detected in interphase nuclei (
apparently in the vicinity of the metaphase plate (
may be cell cycleregulated, but the significance of the AKAP95RII
complex at mitosis remains unknown.
We demonstrate here in in vivo and in vitro immunoblocking and rescue experiments the formation of an AKAP95PKA signaling complex onto mitotic chromosomes, and a role of AKAP95 in chromatin condensation and maintenance of condensed chromosomes during mitosis. The latter process also requires cAMP/PKA signaling and anchoring of PKA to chromatin by AKAP95. The data also suggest that one function of AKAP95 is to promote the recruitment of components of the condensin complex onto chromatin. The results argue towards a critical role of AKAP95 in the regulation of chromatin structure at mitosis and provide a functional significance for increasing PKA activity during mitosis.
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Materials and Methods |
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Buffers, Reagents, and Antibodies
Nuclear isolation buffer (buffer N) consisted of 10 mM Hepes, pH 7.5, 2 mM MgCl2, 250 mM sucrose, 25 mM KCl, 1 mM DTT, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A. Cell lysis buffer consisted of 20 mM Hepes, pH 8.2, 5 mM MgCl2, 10 mM EDTA, 1 mM DTT, and 20 µg/ml cytochalasin B and protease inhibitors. A GST-AKAP95 fragment covering amino acids 387692 and including the RII-binding domain of human AKAP95 (designated GST-AKAP951-386) was described previously (
1-386 fusion peptide. Antihuman RII
mAbs (Transduction Laboratories) were described earlier (
, rabbit antiserum against a peptide of human lamin B, and human antilamin B autoantibodies (gifts from J.-C. Courvalin, Institut Jacques Monod, Paris, France) were described previously (
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Cell Synchronization and Microinjection
HeLa cells were grown in EMEM medium (GIBCO BRL) as described in
For nuclear microinjections, HeLa cells were plated onto glass coverslips at 106 cells per 28-cm2 dish and synchronized in S phase by double thymidine block. Nuclei were microinjected with 2550 pl of solution using a Narishige micromanipulator and picoinjector. Injection solution consisted of EMEM containing 10 µg/ml of a 150-kD FITC-dextran (Sigma Chemical Co.) to visualize the injections. Nuclei were injected with either 25 pg affinity-purified antiAKAP95 polyclonal antibodies (at ~0.1 mg/ml IgG), mAb36 (diluted 1/10), 25 pg preimmune IgGs (at ~0.1 mg/ml), or ~250 pg GST-AKAP951-386 peptide together with antiAKAP95 antibodies. Inhibitors, antagonists, or catalytic peptides were injected at concentrations indicated in Results. Successful nuclear injections were assessed by retention of the FITC-dextran within the nucleus 1 h after injection (see Results). After a resting period of 6 h, injected cells were synchronized in M phase with 1 µM nocodazole. Mitotic cells were microinjected as described above while arrested in M phase with nocodazole. Injected cells remained in nocodazole for up to 2 h after injection during the period of observation.
Mitotic and Interphase Cell Extracts
Mitotic HeLa cells were washed once in ice-cold PBS, once in 20 vol of ice-cold lysis buffer, and packed at 800 g for 10 min. The cell pellet was resuspended in 1 vol of lysis buffer and incubated for 30 min on ice. Cells were homogenized by a 2x 2-min sonication on ice and the lysate was centrifuged at 10,000 g for 15 min at 4°C. The supernatant was cleared at 200,000 g for 3 h at 4°C in a Beckman SW55 rotor. The clear supernatant (mitotic cytosolic extract) was aliquoted, frozen in liquid nitrogen, and stored at -80°C. Protein concentration of the extract was usually ~15 mg/ml. Interphase cytosolic extracts were prepared as above from unsynchronized HeLa cells (9598% in interphase) except that EDTA was omitted from the cell lysis buffer.
Preparation of Nuclei and Chromatin
Confluent unsynchronized HeLa cells were harvested, washed in PBS, sedimented at 400 g, and resuspended in 20 vol of ice-cold buffer N containing 10 µg/ml cytochalasin B. After a 30-min incubation on ice, cells were homogenized on ice by 140170 strokes of a tight fitting (B-type) glass pestle in a 7-ml homogenizer. Nuclei were sedimented at 400 g for 10 min at 4°C and washed twice by resuspending in buffer N and centrifuging as above. Essentially, all nuclei recovered were morphologically intact, as judged by phase-contrast microscopy and labeling with 10 µg/ml FITC-conjugated ConA (data not shown) (
To prepare interphase chromatin, 108 HeLa nuclei suspended in 200 µl buffer N containing 1% Triton X-100 were incubated with 5 U of micrococcal nuclease (Sigma Chemical Co.) at 37°C for 5 min. Digestion was terminated by adding EDTA to 5 mM and the mixture was centrifuged at 10,000 g for 5 min. The supernatant (S1) was collected and the pellet was resuspended in 2 mM EDTA and incubated for 15 min at 4°C. After sedimentation as above, the supernatant (S2) was combined with S1 to yield the chromatin fraction, proteins were precipitated with 10% TCA and dissolved in SDS sample buffer. To solubilize mitotic condensed chromatin, mitotic cell lysates were centrifuged at 10,000 g for 10 min. The pellet was resuspended in 500 µl buffer and sedimented at 1,000 g for 20 min in a swingout rotor (Eppendorf) through a 1-M sucrose cushion made in buffer N. Mitotic chromosomes were recovered from the pellet. This pellet was extracted with 1% Triton X-100 at 4°C for 30 min, sedimented at 15,000 g for 15 min, and the detergent-insoluble fraction was digested with 5 U/ml micrococcal nuclease at 37°C for 5 min. After sedimentation as above, proteins of the supernatant (soluble chromatin) were precipitated in 10% TCA and dissolved in SDS-sample buffer.
Loading of Nuclei with AntiAKAP95 Antibodies
Purified HeLa cell nuclei (2,000 nuclei/µl) were permeabilized in 500 µl buffer N containing 0.75 µg/ml lysolecithin for 15 min at room temperature. Excess lysolecithin was quenched by adding 1 ml of 3% BSA made in buffer N and incubating for 5 min on ice, before sedimenting the nuclei and washing once in buffer N. Nuclei were resuspended at 2,000 nuclei/µl in 100 µl buffer N containing affinity-purified antiAKAP95 antibodies (1:40 dilution; 2.5 µg) or 2.5 µg of preimmune rabbit IgGs. After a 1-h incubation on ice with gentle agitation, nuclei were sedimented at 500 g through 1 M sucrose for 20 min and held in buffer N on ice until use. Antibody loading of nuclei was verified by immunofluorescence. AKAP95 was not proteolyzed during permeabilization and antibody loading procedures, and lysolecithin-treatment of nuclei did not affect nuclear envelope disassembly or chromatin condensation in mitotic extract (data not shown).
Nuclear Disassembly and Chromatin Condensation Assay
A nuclear disassembly/chromatin condensation reaction consisted of 20 µl mitotic extract, 1 µl nuclear suspension (~2 x 104 nuclei), and 0.6 µl of an ATP-generating system (1 mM ATP, 10 mM creatine phosphate, 25 µg/ml creatine kinase;
Premature Chromatin Decondensation Assay
HeLa chromatin condensed in mitotic extract was recovered by sedimentation at 1,000 g through 1 M sucrose, washed by resuspension and sedimentation in lysis buffer, and incubated for up to 2 h in fresh mitotic extract, either intact or immunodepleted of AKAP95 or RII. In some experiments, this extract contained antibodies or inhibitors. DNA was labeled with Hoechst and examined as above. Premature chromatin decondensation (PCD) referred to swelling of the chromatin into ovoid or spherical objects, with no discernible chromosomes. Kinetics of PCD was evaluated by measurement of the proportion of chromatin masses undergoing decondensation at regular time intervals.
Histone H1 Kinase Assay
5 µl of HeLa cytosolic extract was mixed with 5 µl of a 2x histone kinase buffer (160 mM ß-glycerophosphate, 20 mM EGTA, 30 mM MgCl2, 2 mM DTT, 20 µg/ml each of aprotinin, leupeptin, and pepstatin A, and 100 nM PKI). The kinase reaction was initiated by addition of 10 µl of a cocktail containing 2.5 mg/ml histone H1, 0.7 mM ATP, and 50 µCi of -[32P]ATP. The reaction was carried out at room temperature for 15 min and stopped by addition of 15 µl of 2x SDS loading buffer and boiling. Proteins were resolved by 15% SDS-PAGE, the gel was dried, and phosphorylation of histone H1 was visualized by autoradiography.
Immunological Procedures
Immunoblotting of nuclei, chromatin, and reaction supernatants was performed as described ( mAb (1:250), anti-Eg7 (1:500), and HRP-conjugated secondary antibodies. Blots were revealed with chloronaphtol/hydrogen peroxide (
mAb, antiAKAP95 polyclonal, and anti-Eg7, each at a 1:50 dilution) from the supernatant at room temperature for 2.5 h, followed by incubation with protein A/G agarose (1:25 dilution) for 1.5 h and centrifugation at 4,000 g for 10 min. Immune complexes were washed three times in IP buffer and proteins were eluted in 2x SDS boiling sample buffer. RII
was also immunoprecipitated from mitotic extract, or chromatin, or reaction supernatant fractions diluted 10x in IP buffer and sonicated (chromatin fraction only). AKAP95 or RII
was also immunodepleted from undiluted cytosolic extracts using affinity-purified antiAKAP95 antibodies or antiRII
mAbs at a 1:50 dilution. For immunofluorescence analysis (
mAb, antilamin B human autoantibody) and secondary antibodies were used at a 1:100 dilution. DNA was stained with 0.1 µg/ml Hoechst 33342. Observations were made on an Olympus AX70 epifluorescence microscope using a 100x objective, and photographs were taken with a Photonic Science CCD camera and OpenLab software (Improvision Co.).
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Results |
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Subcellular Distribution of Human AKAP95 during the Cell Cycle
The subcellular localization of human AKAP95 was examined throughout the HeLa cell cycle. Immunofluorescence analysis using an affinity-purified antiAKAP95 antibody confirmed that AKAP95 was exclusively nuclear in interphase, colocalized with DNA, and was excluded from nucleoli (Figure 1 A). As early as prometaphase and until telophase, AKAP95 staining colocalized with chromosomes over a diffuse cytoplasmic labeling (Figure 1 A). In late telophase/interphase, AKAP95 labeling became excluded from the cytoplasm and restricted to the daughter nuclei (Figure 1 A). Similar results were obtained with two newly developed antiAKAP95 mAbs, mAb36 and mAb47, and in human 293T fibroblasts with all three antibodies (data not shown).
Immunoblotting analysis of AKAP95 revealed similar levels of AKAP95 in whole interphase and mitotic cell lysates (Figure 1 B). Fractionation of interphase and mitotic cells confirmed that AKAP95 was exclusively nuclear in interphase (Figure 1 C). At mitosis, ~75% of AKAP95 cofractionated with chromatin, whereas ~25% was detected in a 200,000-g supernatant fraction (Figure 1 C, cytosol). No AKAP95 was detected in membrane fractions of interphase or mitotic cells (Figure 1 C). Furthermore, fractionation of purified HeLa nuclei into chromatin and high saltextracted nuclear matrices revealed that ~80% of AKAP95 partitioned with the matrix, whereas ~20% cofractionated with chromatin (Figure 1 D). Thus, AKAP95 is restricted to the nuclei of interphase cells, where it associates primarily with the nuclear matrix. At mitosis, AKAP95 redistributes into a major chromatin-associated fraction and a minor soluble pool. Upon nuclear reassembly at the end of mitosis, AKAP95 is sequestered into the daughter nuclei where it re-acquires an interphase distribution.
AKAP95 Associates with the RII Regulatory Subunit of PKA at Mitosis and in the Mitotic Extract
Association of AKAP95 with RII in interphase and mitosis was investigated in dual immunofluorescence and immunoprecipitation experiments. In interphase, AKAP95 labeling was clearly distinct from the cytoplasmic staining of RII
(Figure 2 A, right cell in all panels). At mitosis, RII
exhibited a more uniform cytoplasmic labeling that mostly overlapped with that of AKAP95 (Figure 2 A, left cell in all panels). Although both AKAP95 and RII
were detected by immunoblotting in interphase whole cell lysates (Figure 2 B, left), immunoprecipitation of RII
from such lysates did not coprecipitate AKAP95 in interphase, but did coprecipitate AKAP95 at mitosis (Figure 2 B, right). Immunoprecipitation of RII
also coprecipitated AKAP95 from both a mitotic cytosolic fraction and from mitotic chromatin solubilized by micrococcal nuclease digestion (Figure 2 C). This illustrates the assembly of both soluble and chromatin-associated AKAP95RII
complexes at mitosis.
The dynamics of formation of the chromatin-associated mitotic AKAP95RII complex was investigated using a cytosolic extract prepared from mitotic HeLa cells. The extract supports disassembly of exogenous purified interphase HeLa nuclei, including nuclear envelope breakdown and chromatin condensation (see Materials and Methods). Immunofluorescence and immunoblotting analysis of input nuclei and condensed chromatin showed that AKAP95 was associated with the condensing chromatin (Figure 3 A,
-AKAP95, and B, top panel). As in vivo, a minor portion of AKAP95 was also released into the cytosol during chromatin condensation (data not shown). Concomitantly, RII
, undetected in input interphase nuclei, was recruited from the extract onto the chromatin surface where it colocalized and cofractionated with AKAP95 (Figure 3 A,
-RII
and Merge, and B, bottom panel). Furthermore, immunoprecipitation of RII
from condensed chromatin coprecipitated AKAP95 (Figure 3 C), illustrating the formation of a chromatin-associated AKAP95RII
complex during chromatin condensation in vitro. It is noteworthy that association of AKAP95 with chromatin also occurred in mitotic extract immunodepleted of RII
(data not shown), indicating that mitotic redistribution of AKAP95 is independent of RII
.
Altogether, these results indicate that in mitotic extract, nuclear AKAP95 is redistributed and primarily associates with condensing chromosomes in an RII-independent manner. Concomitantly, AKAP95 recruits RII
from a cytosolic pool onto chromatin. Thus, one function of chromatin-associated AKAP95 at mitosis is to target PKA, via RII
, to condensing chromosomes.
Mitotic Chromosome Condensation Requires Functional AKAP95
The functional significance of the chromatin-associated AKAP95RII complex was first investigated using the mitotic extract. To this end, we attempted to block the function of nuclear AKAP95 by introducing affinity-purified antiAKAP95 antibodies (or control preimmune rabbit IgGs) into purified HeLa nuclei as described in Materials and Methods. Antibody-loaded nuclei (Figure 4 A, Input) were exposed to mitotic extract and nuclear disassembly assessed after 2 h by DNA staining and immunofluorescence analysis using antilamin B and antiLBR antibodies. Figure 4 A shows that antiAKAP95 antibodies, but not preimmune IgGs, inhibited chromatin condensation although nuclear envelope disassembly took place as judged by the absence of lamin B or LBR labeling. Inhibition of chromatin condensation occurred with most (~90%) chromatin masses examined (see Figure 4 C, Poly) and was reproduced by mAb36, whereas mAb47 was little effective (Figure 4 C). Inhibition of chromatin condensation was specific for antiAKAP95 antibodies since it did not occur with the following: antiAKAP95 antibodies introduced into nuclei with 0.1 mg/ml of the GST-AKAP95
1-386 peptide (Figure 4 B) used to generate the antibodies (Figure 4 C, Poly + AKAP95, mAb36 + AKAP95); and affinity-purified polyclonal antibodies against heterochromatin proteins HP1
(Figure 4 C), HP1ß, or HP1
(not shown) (
Preincubation of antiAKAP95 antibodies in the extract or immunodepletion of soluble AKAP95 did not inhibit condensation (Figure 4 C). This illustrates a role for nuclear rather than cytosolic AKAP95 in chromatin condensation. Additionally, loading nuclei with antiAKAP95 antibodies did not block the recruitment of RII onto the chromatin, as judged by immunofluorescence and immunoblotting analyses of condensed chromatin (data not shown). This argues that inhibition of chromatin condensation with antiAKAP95 antibodies does not result from inhibition of the PKA-anchoring activity of AKAP95.
To determine whether inhibiting nuclear AKAP95 function in vivo affected mitotic chromosome condensation, nuclei of HeLa cells synchronized in S phase by a double thymidine block were microinjected with ~25 pg of antiAKAP95 polyclonal antibodies or mAb36, each with or without 250 pg of competitor GST-AKAP951-386 peptide, or with preimmune IgGs. Successful nuclear injections were verified by coinjection of a 150-kD FITC-dextran and scored by retention of the dextran within the nuclei 1 h after injection (Figure 5 A, a, green). Injected cells were released from the thymidine block, exposed to 1 µM nocodazole, and the proportion of cells arrested at mitosis was determined after 17 h. A fraction of injected cells was also analyzed by immunofluorescence using antibodies against LBR, a marker of the inner nuclear membrane. Figure 5 shows that, after nocodazole treatment, cells injected with antiAKAP95 antibodies did not undergo chromosome condensation, as shown by the absence of detectable metaphase chromosomes under bright field microscopy (Figure 5 A, c and d) and after DNA staining in most cells (Figure 5 C), and by mitotic indexes of 1520% (Figure 5 B). In contrast, preimmune IgGs or antiAKAP95 antibodies injected with GST-AKAP95
1-386 did not prevent chromosomes from forming a metaphase plate (Figure 5 A, b and e, and B; see Table 1). In addition, despite the lack of chromosome condensation in antibody-injected cells, the release of FITC-dextran from the nucleus into the cytoplasm of these cells after nocodazole treatment (Figure 5 A, c and d, green) argued that the nuclear envelope was disassembled. This was verified by immunofluorescence analysis of LBR (Figure 5 C). Breakdown of the nuclear envelope implies that immunoblocking of nuclear AKAP95 does not prevent entry into mitosis per se, but rather affects chromosome structure at this stage of the cell cycle. However, it should be noted that microinjection of a 25-fold higher concentration of antiAKAP95 antibodies blocked nuclear envelope breakdown in the majority of injected cells (data not shown).
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Mitotic Chromosome Condensation Does Not Require Anchoring of PKA to AKAP95 nor PKA Activity
The recruitment of RII by AKAP95 onto condensing chromatin led us to determine whether the putative role of AKAP95 in chromatin condensation required association with RII
and anchoring of PKA. To this end, the RII-anchoring inhibitor peptide Ht31 (500 nM) was preincubated in mitotic extract before adding nuclei. Chromatin condensed to the same extent with Ht31 and control Ht31-P peptides that do not compete binding of RII to AKAPs (data not shown). Thus, the role of AKAP95 in chromosome condensation in vitro is independent of its RII-anchoring function. Furthermore, neither chromatin condensation nor nuclear envelope disassembly was affected by inhibiting PKA activity in the extract with 1 µM of the PKA inhibitor PKI, or by downregulating cAMP signaling with 100 µM of the cAMP antagonists Rp-8-Br-cAMPS or Rp-8-CPT-cAMPS (data not shown). Thus, chromosome condensation in mitotic extract does not require cAMP signaling or PKA activity.
The effects of disrupting AKAP-RII anchoring and downregulating cAMP/PKA signaling on chromosome condensation at mitosis were investigated by microinjecting nuclei of interphase HeLa cells with 50 nM Ht31, 50 nM Ht31-P, 10 nM PKI, or 10 µM Rp-8-Br-cAMPS, and assessing mitotic indexes after nocodazole treatment as done previously after immunoblocking of AKAP95. As shown in Table 1, neither reagent inhibited nuclear envelope breakdown (data not shown) nor chromosome condensation, as judged by mitotic indexes of 7990%. Therefore, disruption of AKAP95-RII anchoring or cAMP/PKA inhibition does not impair mitotic chromosome condensation.
AKAP95 Function Is Required to Maintain Chromosomes Condensed during Mitosis
Although chromatin condensation occurred normally in the mitotic extract containing antiAKAP95 antibodies, we consistently observed a phase of decondensation of the chromatin upon prolonged (>4 h) incubation in the extract (data not shown). This raised the hypothesis that inhibition of AKAP95 might affect the morphology of the condensed chromatin. To test this possibility, chromatin was allowed to condense for 2 h in mitotic extract, antiAKAP95 antibodies were added (1:50 dilution) and chromatin morphology was examined by DNA staining after another 2 h of incubation. Chromatin decondensation was observed in extract containing antiAKAP95 antibodies (Figure 6 A, +-AKAP95), but not in the extract containing preimmune IgGs or antiAKAP95 antibodies together with 0.1 mg/ml of GST-AKAP95
1-386 competitor peptide (Figure 6 A). Thus, immunoblocking AKAP95 in the extract prevented the chromatin from remaining in a condensed form. To determine whether immunodepletion of AKAP95 had a similar effect, chromatin condensed in mitotic extract was purified by sedimentation and further incubated in a fresh extract immunodepleted of AKAP95. Chromatin exposed to AKAP95-depleted, but not mock-depleted, extract underwent decondensation within 2 h (Figure 6 A), indicating that AKAP95 is implicated in maintaining chromatin in a condensed form. Moreover, the extent of chromatin decondensation after immunoblocking or immunodepletion of AKAP95 was more limited than decondensation of mitotic chromatin exposed to an interphase extract (Figure 6 A, Interphase), suggesting the involvement of distinct decondensation pathways. Consequently, chromatin decondensation in mitotic extract was referred to as premature chromatin decondensation (PCD).
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As PCD might be interpreted as a consequence of premature exit of the extract from the M phase, we tested this possibility by measuring the level of histone H1 kinase activity in the mitotic extract incubated for 2 h without or with antiAKAP95 antibodies and in AKAP95-depleted extract. Figure 6 B shows that elevated (mitotic) levels of H1 kinase activity were maintained in antibody-containing and immunodepleted extracts (compare with basal H1 kinase activity in interphase HeLa cell extract). This result indicates that immunoblocking or immunodepleting AKAP95 did not release the extract from the M phase, and lends further support to the hypothesis that PCD and interphase chromatin decondensation involve distinct processes.
Conclusive evidence that AKAP95 was required for maintenance of condensed chromatin in vitro was provided in a rescue experiment. Purified condensed chromatin was exposed to mitotic extract immunodepleted of AKAP95. Increasing concentrations of GST-AKAP951-386 were added and after 2 h proportions of PCD were determined by DNA staining. GST-AKAP95
1-386 dramatically inhibited PCD in a concentration-dependent manner (Figure 6 C). We determined whether GST-AKAP95
1-386 was also able to promote recondensation of prematurely decondensed chromatin. Purified condensed chromatin was allowed to undergo PCD for 2 h in AKAP95-depleted mitotic extract. Subsequent addition of GST-AKAP95
1-386 to the extract restored chromatin condensation also in a concentration-dependent manner (Figure 6 D). Thus, GST-AKAP95
1-386 was capable of inhibiting PCD and recondensing prematurely decondensed chromatin in AKAP95-depleted extract, indicating that AKAP95 is required for maintaining chromatin in a condensed form. Furthermore, since antiAKAP95 antibodies do not block binding of RII
to AKAP95, the data also argue that AKAP95 has an intrinsic function in the maintenance of condensed chromatin.
The relevance of these in vitro observations on chromosome structure during mitosis was investigated. HeLa cells arrested in the M phase with 1 µM nocodazole were injected with affinity-purified antiAKAP95 antibodies and, after 1 h, chromatin morphology was assessed by phase-contrast and DNA staining while cells remained in nocodazole. AntiAKAP95 antibodies readily induced decondensation of chromosomes that could no longer be distinguished by phase-contrast (Figure 7, -AKAP95, arrow points to the metaphase chromosomes of a mitotic cell that was not injected). Furthermore, no nuclear envelope was detected, arguing that decondensation was reminiscent of PCD observed previously in vitro and distinct from interphase nuclear reformation. In contrast, coinjection of antiAKAP95 antibodies with 250 pg GST-AKAP95
1-386 did not alter the state of chromosome condensation (Figure 7,
-AKAP95 + pept.). These results indicate that chromosome decondensation is not an artefactual consequence of exposure of cells to nocodazole. Rather, they show that as in in vitro, immunoblocking AKAP95 at mitosis induces PCD, indicating that functional AKAP95 is needed for maintaining chromosomes condensed throughout mitosis.
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Anchoring of RII to AKAP95 and PKA Activity Are Required for Maintenance of Condensed Chromatin during Mitosis
Whether AKAP95RII association and cAMP signaling through PKA were required for maintenance of condensed chromatin during M phase was examined. Purified condensed chromatin was added to a mitotic extract preincubated with 500 nM Ht31 (or control Ht31-P) peptides to disrupt AKAP95RII interactions (data not shown) and PCD was assessed after 1 h by DNA staining. Figure 8 A shows that while chromatin remained condensed with Ht31-P, Ht31 induced PCD, indicating a requirement for AKAP95RII interaction to maintain chromatin condensed. Whether PKA activity and cAMP signaling were involved in this process was determined using PKA inhibitor PKI (1 µM) and the cAMP antagonist Rp-8-Br-cAMPS (100 µM). Both reagents induced PCD, whereas control activation of PKA with 1 µM cAMP had no effect (Figure 8 A). Adding free C subunits together with antiAKAP95 antibodies also induced PCD (Figure 8 A), suggesting that only PKA bound to chromatin-associated AKAP95 is implicated in maintaining condensed chromatin. Altogether, these results indicate that maintenance of condensed chromatin in mitotic extract requires functional AKAP95, cAMP signaling events mediated by PKA and anchoring of PKA (via RII
) to chromosomes by AKAP95.
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The requirement for RII binding to AKAP95 and PKA activity for maintenance of condensed chromatin was tested further by assessing chromatin morphology after immunodepleting RII
from the extract. Up to 60% of condensed chromatin masses incubated in the mitotic extract depleted of RII
underwent PCD over a 2-h period (Figure 8 B). Moreover, immunoblotting analysis of chromatin at the beginning (0 h) and at the end (120 min) of incubation in an RII
-depleted extract showed that whereas input chromatin harbored RII
, most RII
was solubilized by 120 min (Figure 8 C, bottom). In contrast, no obvious change in the amount of AKAP95 bound to chromatin was detected under these conditions (Figure 8 C, top). Together with our previous observation that RII
is recruited from the cytosol to the chromatin via AKAP95, these results reflect a dissociation of RII
from the chromatin and suggest that, as with AKAP95, keeping RII
on condensed chromatin involves an equilibrium between soluble and chromatin-associated pools of PKA.
The requirements for AKAPRII interaction and cAMP/PKA signaling events to maintain chromosomes condensed at mitosis were investigated by microinjecting M phasearrested HeLa cells with either 50 nM Ht31 or Ht31-P, 10 nM PKI, 10 µM Rp-8-Br-cAMPS, ~1 ng C, or 25 pg anti-AKAP95 antibodies together with ~1 ng C. The cells remained exposed to nocodazole for 1 h after injections and were examined by phase-contrast microscopy and DNA staining. Data of Figure 9 show that Ht31 (but not Ht31-P), PKI and Rp-8-Br-cAMPS induced PCD, indicating that PKA-AKAP anchoring, PKA activity, and cAMP signaling are required for maintenance of condensed chromatin throughout mitosis. Coinjection of antiAKAP95 antibodies with C also promoted PCD (Figure 9), arguing that functional inhibition of AKAP95 promotes chromatin decondensation even in the presence of unbound PKA.
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Immunoblocking of AKAP95 Inhibits Recruitment of Eg7 to Chromatin in Mitotic Extract
Several proteins have been shown to be required for mitotic chromosome condensation, including pEg7 (
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pEg7, the Xenopus homologue of human Eg7, has been previously shown to be recruited onto condensing chromosomes at mitosis (-AKAP95) or antiAKAP95 antibodies (+
-AKAP95) were allowed to condense for 2 h in mitotic extract. Chromatin masses were sedimented and immunoblotted using antiEg7 antibodies. As expected from our previous results antiAKAP95 antibodies blocked chromatin condensation. Remarkably, the antibodies also prevented the recruitment of Eg7 to chromatin (Figure 10 E, +
-AKAP95) that normally occurs in extract either in the absence of antibodies (not shown) or with nuclei loaded with preimmune IgGs (Figure 10 D, -
-AKAP95). Loading nuclei with antiHP1
antibodies did not impede association of Eg7 with chromatin (Figure 10 E, +
-HP1
), indicating that inhibition of Eg7 recruitment by antiAKAP95 antibodies is not a mere consequence of steric hindrance imposed by the antibody. Altogether, these results suggest that functional inhibition of AKAP95 prevents the association of Eg7 to chromatin required for condensation.
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Discussion |
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This paper provides evidence that AKAP95 is a multivalent protein with a role in chromosome condensation at mitosis. AKAP95 acts as an anchor for a PKA-signaling complex onto mitotic chromosomes, which is required for maintenance of chromosomes in a condensed form throughout mitosis. AKAP95 also appears essential for the recruitment onto chromosomes of Eg7, a component of the condensin complex. The data also provide insights on the significance of the rise in cAMP level and PKA activity during mitosis.
Several lines of evidence illustrate a function of AKAP95 in chromosome condensation and maintenance of chromosomes in a condensed form during mitosis. First, immunoblocking intranuclear AKAP95 inhibits chromatin condensation in vitro and in vivo. Second, immunoblocking AKAP95 during mitosis or in vitro, or immunodepletion of soluble AKAP95 from the mitotic extract, induces PCD. Inhibition of chromatin condensation with antiAKAP95 antibodies is unlikely to result from a nonspecific steric effect of the antibodies since antibodies against all variants of the heterochromatin protein HP1 (
A third observation supporting the involvement of AKAP95 in regulating chromosome structure at mitosis is the finding that a recombinant AKAP95 fragment (GST-AKAP951-386) prevents PCD in AKAP95-depleted extract, and restores condensation of prematurely decondensed chromatin, both in a dose-dependent manner. Thus, the domain(s) of AKAP95 required for this activity and for interaction of AKAP95 with chromatin reside(s) in the carboxy-terminal 306 amino acids (amino acids 387692) of the protein. As expected from the RII binding requirement for maintenance of condensed chromatin, this AKAP95 fragment also contains the RII binding motif (
The consequences of immunoblocking AKAP95 may be accounted for by the hypothesis that AKAP95 function may be disrupted by antibodies only when they bind AKAP95 that is not associated with chromatin. Indeed, the antibodies block condensation when incorporated into interphase nuclei before association of AKAP95 with chromatin (Landsverk, H., and P. Collas, unpublished results). Thus, it is possible that antiAKAP95 antibodies incubated in mitotic cytosol fail to inhibit chromatin condensation because by the time the nuclear envelope breaks down, nuclear AKAP95 has already associated with chromosomes. Similarly, induction of PCD in cytosol immunodepleted of AKAP95 suggests that the establishment of an equilibrium between chromosome-associated and soluble AKAP95 is critical for maintenance of condensation. By binding AKAP95 when it dissociates from chromosomes as well as soluble AKAP95, the antibodies may sharply decrease the on-rate of AKAP95 to chromosomes, causing decondensation of the chromatin.
Since it associates with chromatin early during mitotic nuclear disassembly, AKAP95 could conceivably either directly affect chromatin structure, or facilitate the recruitment of factors controlling mitotic chromosome condensation (
The multivalent nature of AKAP95 is not only evidenced by its role in chromatin condensation, but also by the observation that AKAP95 maintains a crucial PKA-anchoring function during mitosis. Whereas AKAP95 and RII are found in a different compartment in interphase, a chromosome-associated AKAP95PKA signaling complex is established at mitosis. Induction of PCD after immunodepletion of RII from mitotic cytosol reflects an equilibrium between chromatin-associated (via AKAP95) and soluble RII
, and a requirement for a soluble pool of RII
to maintain the chromatin fraction of the AKAP95RII
complex saturated and functional. Disruption of PKA anchoring with Ht31 peptides induces PCD in vitro and in vivo, indicating that the AKAP95PKA interaction is essential for the maintenance of condensed chromatin. Several physiological functions implicating AKAP-anchored PKA have been reported using similar disruption approaches (
Our results provide some significance for the rising level of cAMP and increasing PKA activity during mitosis (
Although the structural determinants mediating AKAPRII interactions in general are being characterized ( is primarily localized in the centrosomeGolgi area (
from centrosomes at mitosis correlates with CDK1-mediated phosphorylation of RII
(
in vitro is sufficient to induce partial solubilization of RII
from a Triton X-100 insoluble pool (
is mitosis-specific. Finally, decondensation of in vitrocondensed chromatin in an interphase extract is associated with release of RII
from chromatin-associated AKAP95 (Collas, P., data not shown). This argues that binding of RII
to AKAP95 at mitosis is not a mere consequence of both proteins being in the same subcellular compartment; rather, a modification of either protein seems to affect their interaction. Together with the data of
, suggesting an additional mechanism regulating PKA type II subcellular localization.
An emerging feature of AKAPs is their ability to anchor entire signaling complexes other than PKA to specific substrates. To date, AKAP79, AKAP220, gravin, yotiao/AKAP450/CG-NAP, and ezrin have been shown to act as polyvalent anchoring proteins for signaling units involving PKA or protein kinase C together with protein phosphatases (
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Footnotes |
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1 Abbreviations used in this paper: AKAP, A-kinaseanchoring protein; CDK1, cyclin-dependent kinase 1; LBR, lamin B receptor; PCD, premature chromatin decondensation; PKA, protein kinase A; R and C, regulatory and catalytic subunit of PKA, respectively; SMC, structural maintenance of chromosome.
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Acknowledgements |
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We are thankful to J.-C. Courvalin for gifts of antilamin B, antiLBR, and antiHP1 antibodies, T. Lømo (Institute of Neurophysiology, University of Oslo) for use of his photography facility, and K.A. Tasken for critical reading of the manuscript and discussions.
This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council, the Anders Jahre Foundation, the Novo Nordisk Foundation Committee (to P. Collas and K. Tasken), and Association pour la Recherche contre le Cancer (No. B09808; to K. Le Guellec). P. Collas and K. Taskén are fellows of the Norwegian Cancer Society.
Submitted: 27 August 1999
Revised: 18 October 1999
Accepted: 3 November 1999
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References |
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