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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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Subcellular and subnuclear localization of AKAP95 in HeLa cells. (A) The distribution of AKAP95 during the HeLa cell cycle was examined by immunofluorescence analysis of unsynchronized cells using an affinity-purified polyclonal anti–AKAP95 antibody. DNA (insets) was labeled with Hoechst 33342. Bar, 10 μm. (B) Interphase (I) and mitotic (M) HeLa cells (107 cells each) were dissolved in SDS-sample buffer, proteins separated by SDS-PAGE, and immunoblotted using the affinity-purified anti–AKAP95 antibody. (C) Interphase and mitotic HeLa cells (107 and 5 × 107, respectively) were fractionated into nuclei or chromatin, cytosol, and cytoplasmic membranes, and proteins of each fraction were immunoblotted using anti–AKAP95 antibodies. Relative amounts of AKAP95 in each fraction were determined by densitometric analysis of duplicate blots. (D) Purified HeLa nuclei (108) were fractionated into chromatin and high salt–extracted nuclear matrices, proteins were immunoblotted using anti–AKAP95 antibodies, and duplicate blots analyzed by densitometry.
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Figure 1: Subcellular and subnuclear localization of AKAP95 in HeLa cells. (A) The distribution of AKAP95 during the HeLa cell cycle was examined by immunofluorescence analysis of unsynchronized cells using an affinity-purified polyclonal anti–AKAP95 antibody. DNA (insets) was labeled with Hoechst 33342. Bar, 10 μm. (B) Interphase (I) and mitotic (M) HeLa cells (107 cells each) were dissolved in SDS-sample buffer, proteins separated by SDS-PAGE, and immunoblotted using the affinity-purified anti–AKAP95 antibody. (C) Interphase and mitotic HeLa cells (107 and 5 × 107, respectively) were fractionated into nuclei or chromatin, cytosol, and cytoplasmic membranes, and proteins of each fraction were immunoblotted using anti–AKAP95 antibodies. Relative amounts of AKAP95 in each fraction were determined by densitometric analysis of duplicate blots. (D) Purified HeLa nuclei (108) were fractionated into chromatin and high salt–extracted nuclear matrices, proteins were immunoblotted using anti–AKAP95 antibodies, and duplicate blots analyzed by densitometry.

Mentions: The subcellular localization of human AKAP95 was examined throughout the HeLa cell cycle. Immunofluorescence analysis using an affinity-purified anti–AKAP95 antibody confirmed that AKAP95 was exclusively nuclear in interphase, colocalized with DNA, and was excluded from nucleoli (Fig. 1 A). As early as prometaphase and until telophase, AKAP95 staining colocalized with chromosomes over a diffuse cytoplasmic labeling (Fig. 1 A). In late telophase/interphase, AKAP95 labeling became excluded from the cytoplasm and restricted to the daughter nuclei (Fig. 1 A). Similar results were obtained with two newly developed anti–AKAP95 mAbs, mAb36 and mAb47, and in human 293T fibroblasts with all three antibodies (data not shown).


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)

Subcellular and subnuclear localization of AKAP95 in HeLa cells. (A) The distribution of AKAP95 during the HeLa cell cycle was examined by immunofluorescence analysis of unsynchronized cells using an affinity-purified polyclonal anti–AKAP95 antibody. DNA (insets) was labeled with Hoechst 33342. Bar, 10 μm. (B) Interphase (I) and mitotic (M) HeLa cells (107 cells each) were dissolved in SDS-sample buffer, proteins separated by SDS-PAGE, and immunoblotted using the affinity-purified anti–AKAP95 antibody. (C) Interphase and mitotic HeLa cells (107 and 5 × 107, respectively) were fractionated into nuclei or chromatin, cytosol, and cytoplasmic membranes, and proteins of each fraction were immunoblotted using anti–AKAP95 antibodies. Relative amounts of AKAP95 in each fraction were determined by densitometric analysis of duplicate blots. (D) Purified HeLa nuclei (108) were fractionated into chromatin and high salt–extracted nuclear matrices, proteins were immunoblotted using anti–AKAP95 antibodies, and duplicate blots analyzed by densitometry.
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Figure 1: Subcellular and subnuclear localization of AKAP95 in HeLa cells. (A) The distribution of AKAP95 during the HeLa cell cycle was examined by immunofluorescence analysis of unsynchronized cells using an affinity-purified polyclonal anti–AKAP95 antibody. DNA (insets) was labeled with Hoechst 33342. Bar, 10 μm. (B) Interphase (I) and mitotic (M) HeLa cells (107 cells each) were dissolved in SDS-sample buffer, proteins separated by SDS-PAGE, and immunoblotted using the affinity-purified anti–AKAP95 antibody. (C) Interphase and mitotic HeLa cells (107 and 5 × 107, respectively) were fractionated into nuclei or chromatin, cytosol, and cytoplasmic membranes, and proteins of each fraction were immunoblotted using anti–AKAP95 antibodies. Relative amounts of AKAP95 in each fraction were determined by densitometric analysis of duplicate blots. (D) Purified HeLa nuclei (108) were fractionated into chromatin and high salt–extracted nuclear matrices, proteins were immunoblotted using anti–AKAP95 antibodies, and duplicate blots analyzed by densitometry.
Mentions: The subcellular localization of human AKAP95 was examined throughout the HeLa cell cycle. Immunofluorescence analysis using an affinity-purified anti–AKAP95 antibody confirmed that AKAP95 was exclusively nuclear in interphase, colocalized with DNA, and was excluded from nucleoli (Fig. 1 A). As early as prometaphase and until telophase, AKAP95 staining colocalized with chromosomes over a diffuse cytoplasmic labeling (Fig. 1 A). In late telophase/interphase, AKAP95 labeling became excluded from the cytoplasm and restricted to the daughter nuclei (Fig. 1 A). Similar results were obtained with two newly developed anti–AKAP95 mAbs, mAb36 and mAb47, and in human 293T fibroblasts with all three antibodies (data not shown).

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