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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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Disruption of AKAP95–RII interaction and downregulation of cAMP/PKA signaling promote PCD in mitotic cells. Mitotic HeLa cells were injected as in Fig. 7 with either 50 nM RII-anchoring inhibitor peptide Ht31, 50 nM control Ht31-P, 10 nM PKI, 10 μM cAMP antagonist Rp-8-Br-cAMPS, ∼1 ng C, or ∼1 ng C together with 2–5 pg anti–AKAP95 antibodies. Cells remained in nocodazole after injection and were examined as in Fig. 7. Percentage of PCD was calculated from 30–40 injected cells. Bars, 10 μm.
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Figure 9: Disruption of AKAP95–RII interaction and downregulation of cAMP/PKA signaling promote PCD in mitotic cells. Mitotic HeLa cells were injected as in Fig. 7 with either 50 nM RII-anchoring inhibitor peptide Ht31, 50 nM control Ht31-P, 10 nM PKI, 10 μM cAMP antagonist Rp-8-Br-cAMPS, ∼1 ng C, or ∼1 ng C together with 2–5 pg anti–AKAP95 antibodies. Cells remained in nocodazole after injection and were examined as in Fig. 7. Percentage of PCD was calculated from 30–40 injected cells. Bars, 10 μm.

Mentions: The requirements for AKAP–RII interaction and cAMP/PKA signaling events to maintain chromosomes condensed at mitosis were investigated by microinjecting M phase–arrested HeLa cells with either 50 nM Ht31 or Ht31-P, 10 nM PKI, 10 μM Rp-8-Br-cAMPS, ∼1 ng C, or 2–5 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 Fig. 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 anti–AKAP95 antibodies with C also promoted PCD (Fig. 9), arguing that functional inhibition of AKAP95 promotes chromatin decondensation even in the presence of unbound PKA.


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)

Disruption of AKAP95–RII interaction and downregulation of cAMP/PKA signaling promote PCD in mitotic cells. Mitotic HeLa cells were injected as in Fig. 7 with either 50 nM RII-anchoring inhibitor peptide Ht31, 50 nM control Ht31-P, 10 nM PKI, 10 μM cAMP antagonist Rp-8-Br-cAMPS, ∼1 ng C, or ∼1 ng C together with 2–5 pg anti–AKAP95 antibodies. Cells remained in nocodazole after injection and were examined as in Fig. 7. Percentage of PCD was calculated from 30–40 injected cells. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168084&req=5

Figure 9: Disruption of AKAP95–RII interaction and downregulation of cAMP/PKA signaling promote PCD in mitotic cells. Mitotic HeLa cells were injected as in Fig. 7 with either 50 nM RII-anchoring inhibitor peptide Ht31, 50 nM control Ht31-P, 10 nM PKI, 10 μM cAMP antagonist Rp-8-Br-cAMPS, ∼1 ng C, or ∼1 ng C together with 2–5 pg anti–AKAP95 antibodies. Cells remained in nocodazole after injection and were examined as in Fig. 7. Percentage of PCD was calculated from 30–40 injected cells. Bars, 10 μm.
Mentions: The requirements for AKAP–RII interaction and cAMP/PKA signaling events to maintain chromosomes condensed at mitosis were investigated by microinjecting M phase–arrested HeLa cells with either 50 nM Ht31 or Ht31-P, 10 nM PKI, 10 μM Rp-8-Br-cAMPS, ∼1 ng C, or 2–5 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 Fig. 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 anti–AKAP95 antibodies with C also promoted PCD (Fig. 9), arguing that functional inhibition of AKAP95 promotes chromatin decondensation even in the presence of unbound PKA.

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