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The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis.

Collas P, Le Guellec K, Taskén K - J. Cell Biol. (1999)

Bottom Line: 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.We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, Blindern, 0317 Oslo, Norway. philippe.collas@basalmed.uio.no

ABSTRACT
Protein kinase A (PKA) and the nuclear A-kinase-anchoring 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 AKAP95-PKA 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 RIIalpha 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 306-carboxy-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/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.

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Induction of PCD in mitotic HeLa cells by immunoblocking of AKAP95. HeLa cells were synchronized in metaphase by thymidine block and nocodazole treatment. Mitotic cells were injected with either 2–5 pg affinity-purified polyclonal anti–AKAP95 antibodies or anti–AKAP95 antibodies together with 250 pg competitor GST-AKAP95Δ1-386 peptide. Control cells were injected with FITC-dextran only (top). Cells remained in nocodazole after injection and were examined after 1 h by phase-contrast and fluorescence (FITC) microscopy. DNA was labeled with Hoechst 33342. Percentage of PCD was calculated from 30–40 cells injected per treatment. Arrow points to a noninjected cell that did not undergo PCD. Bars, 10 μm.
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Figure 7: Induction of PCD in mitotic HeLa cells by immunoblocking of AKAP95. HeLa cells were synchronized in metaphase by thymidine block and nocodazole treatment. Mitotic cells were injected with either 2–5 pg affinity-purified polyclonal anti–AKAP95 antibodies or anti–AKAP95 antibodies together with 250 pg competitor GST-AKAP95Δ1-386 peptide. Control cells were injected with FITC-dextran only (top). Cells remained in nocodazole after injection and were examined after 1 h by phase-contrast and fluorescence (FITC) microscopy. DNA was labeled with Hoechst 33342. Percentage of PCD was calculated from 30–40 cells injected per treatment. Arrow points to a noninjected cell that did not undergo PCD. Bars, 10 μm.

Mentions: 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 anti–AKAP95 antibodies and, after 1 h, chromatin morphology was assessed by phase-contrast and DNA staining while cells remained in nocodazole. Anti–AKAP95 antibodies readily induced decondensation of chromosomes that could no longer be distinguished by phase-contrast (Fig. 7, α-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 anti–AKAP95 antibodies with 250 pg GST-AKAP95Δ1-386 did not alter the state of chromosome condensation (Fig. 7, α-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.


The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis.

Collas P, Le Guellec K, Taskén K - J. Cell Biol. (1999)

Induction of PCD in mitotic HeLa cells by immunoblocking of AKAP95. HeLa cells were synchronized in metaphase by thymidine block and nocodazole treatment. Mitotic cells were injected with either 2–5 pg affinity-purified polyclonal anti–AKAP95 antibodies or anti–AKAP95 antibodies together with 250 pg competitor GST-AKAP95Δ1-386 peptide. Control cells were injected with FITC-dextran only (top). Cells remained in nocodazole after injection and were examined after 1 h by phase-contrast and fluorescence (FITC) microscopy. DNA was labeled with Hoechst 33342. Percentage of PCD was calculated from 30–40 cells injected per treatment. Arrow points to a noninjected cell that did not undergo PCD. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 7: Induction of PCD in mitotic HeLa cells by immunoblocking of AKAP95. HeLa cells were synchronized in metaphase by thymidine block and nocodazole treatment. Mitotic cells were injected with either 2–5 pg affinity-purified polyclonal anti–AKAP95 antibodies or anti–AKAP95 antibodies together with 250 pg competitor GST-AKAP95Δ1-386 peptide. Control cells were injected with FITC-dextran only (top). Cells remained in nocodazole after injection and were examined after 1 h by phase-contrast and fluorescence (FITC) microscopy. DNA was labeled with Hoechst 33342. Percentage of PCD was calculated from 30–40 cells injected per treatment. Arrow points to a noninjected cell that did not undergo PCD. Bars, 10 μm.
Mentions: 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 anti–AKAP95 antibodies and, after 1 h, chromatin morphology was assessed by phase-contrast and DNA staining while cells remained in nocodazole. Anti–AKAP95 antibodies readily induced decondensation of chromosomes that could no longer be distinguished by phase-contrast (Fig. 7, α-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 anti–AKAP95 antibodies with 250 pg GST-AKAP95Δ1-386 did not alter the state of chromosome condensation (Fig. 7, α-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.

Bottom Line: 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.We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, Blindern, 0317 Oslo, Norway. philippe.collas@basalmed.uio.no

ABSTRACT
Protein kinase A (PKA) and the nuclear A-kinase-anchoring 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 AKAP95-PKA 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 RIIalpha 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 306-carboxy-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/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.

Show MeSH
Related in: MedlinePlus