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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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AKAP95–RIIα interaction, cAMP signaling, and PKA activity are required for maintenance of condensed chromatin in mitotic extract. (A) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract containing either anti–AKAP95 antibodies (1:50 dilution), 500 nM Ht31, 500 nM Ht31-P, 1 μM PKI, 100 μM Rp-8-Br-cAMPS, 1 μM cAMP, 15 ng/μl recombinant catalytic subunit of PKA (C), or C plus anti–AKAP95 antibodies. Proportions (percent ± SD) of PCD were determined by DNA labeling after 90 min. (B) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract immunodepleted of RIIα. Proportions (percent ± SD) of PCD were determined by DNA staining of sample aliquots at regular intervals. (C) Chromatin fractions at the start (Input) and at the end (120 min) of incubation in RIIα-depleted mitotic extract were sedimented and proteins were immunoblotted using anti–AKAP95 and anti–RIIα antibodies.
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Figure 8: AKAP95–RIIα interaction, cAMP signaling, and PKA activity are required for maintenance of condensed chromatin in mitotic extract. (A) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract containing either anti–AKAP95 antibodies (1:50 dilution), 500 nM Ht31, 500 nM Ht31-P, 1 μM PKI, 100 μM Rp-8-Br-cAMPS, 1 μM cAMP, 15 ng/μl recombinant catalytic subunit of PKA (C), or C plus anti–AKAP95 antibodies. Proportions (percent ± SD) of PCD were determined by DNA labeling after 90 min. (B) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract immunodepleted of RIIα. Proportions (percent ± SD) of PCD were determined by DNA staining of sample aliquots at regular intervals. (C) Chromatin fractions at the start (Input) and at the end (120 min) of incubation in RIIα-depleted mitotic extract were sedimented and proteins were immunoblotted using anti–AKAP95 and anti–RIIα antibodies.

Mentions: Whether AKAP95–RIIα 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 AKAP95–RII interactions (data not shown) and PCD was assessed after 1 h by DNA staining. Fig. 8 A shows that while chromatin remained condensed with Ht31-P, Ht31 induced PCD, indicating a requirement for AKAP95–RII 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 (Fig. 8 A). Adding free C subunits together with anti–AKAP95 antibodies also induced PCD (Fig. 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 RIIα) to chromosomes by AKAP95.


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)

AKAP95–RIIα interaction, cAMP signaling, and PKA activity are required for maintenance of condensed chromatin in mitotic extract. (A) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract containing either anti–AKAP95 antibodies (1:50 dilution), 500 nM Ht31, 500 nM Ht31-P, 1 μM PKI, 100 μM Rp-8-Br-cAMPS, 1 μM cAMP, 15 ng/μl recombinant catalytic subunit of PKA (C), or C plus anti–AKAP95 antibodies. Proportions (percent ± SD) of PCD were determined by DNA labeling after 90 min. (B) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract immunodepleted of RIIα. Proportions (percent ± SD) of PCD were determined by DNA staining of sample aliquots at regular intervals. (C) Chromatin fractions at the start (Input) and at the end (120 min) of incubation in RIIα-depleted mitotic extract were sedimented and proteins were immunoblotted using anti–AKAP95 and anti–RIIα antibodies.
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Related In: Results  -  Collection

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Figure 8: AKAP95–RIIα interaction, cAMP signaling, and PKA activity are required for maintenance of condensed chromatin in mitotic extract. (A) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract containing either anti–AKAP95 antibodies (1:50 dilution), 500 nM Ht31, 500 nM Ht31-P, 1 μM PKI, 100 μM Rp-8-Br-cAMPS, 1 μM cAMP, 15 ng/μl recombinant catalytic subunit of PKA (C), or C plus anti–AKAP95 antibodies. Proportions (percent ± SD) of PCD were determined by DNA labeling after 90 min. (B) Chromatin condensed in mitotic extract was purified and exposed to fresh mitotic extract immunodepleted of RIIα. Proportions (percent ± SD) of PCD were determined by DNA staining of sample aliquots at regular intervals. (C) Chromatin fractions at the start (Input) and at the end (120 min) of incubation in RIIα-depleted mitotic extract were sedimented and proteins were immunoblotted using anti–AKAP95 and anti–RIIα antibodies.
Mentions: Whether AKAP95–RIIα 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 AKAP95–RII interactions (data not shown) and PCD was assessed after 1 h by DNA staining. Fig. 8 A shows that while chromatin remained condensed with Ht31-P, Ht31 induced PCD, indicating a requirement for AKAP95–RII 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 (Fig. 8 A). Adding free C subunits together with anti–AKAP95 antibodies also induced PCD (Fig. 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 RIIα) to chromosomes by AKAP95.

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