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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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Immunoblocking of AKAP95 inhibits the association of Eg7 to condensed chromatin in mitotic extract. (A) Immunoblot of interphase (I) and mitotic (M) HeLa cell lysates with antibodies against Eg7, a component of the human condensin complex. (B) AKAP95 and Eg7 were immunoprecipitated from interphase HeLa cell lysates and precipitates were immunoblotted with each precipitating antibody. (C) AKAP95 and Eg7 were immunoprecipitated from mitotic HeLa cell lysates and precipitates immunoblotted as in B. (D) AKAP95 and Eg7 were immunoprecipitated from mitotic chromatin after solubilization with micrococcal nuclease and digestion with DNase I. Precipitates were immunoblotted as in B. (B–D) Control immunoprecipitations were carried out with preimmune rabbit IgGs (IgG). (E) Interphase nuclei (N) loaded with preimmune IgGs (−α-AKAP95), anti–AKAP95 antibodies (+α-AKAP95) or anti–HP1α antibodies (+α-HP1α) were allowed to condense in mitotic extract. After 2 h, chromatin masses (Ch) were sedimented and immunoblotted using anti–Eg7 antibodies.
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Figure 10: Immunoblocking of AKAP95 inhibits the association of Eg7 to condensed chromatin in mitotic extract. (A) Immunoblot of interphase (I) and mitotic (M) HeLa cell lysates with antibodies against Eg7, a component of the human condensin complex. (B) AKAP95 and Eg7 were immunoprecipitated from interphase HeLa cell lysates and precipitates were immunoblotted with each precipitating antibody. (C) AKAP95 and Eg7 were immunoprecipitated from mitotic HeLa cell lysates and precipitates immunoblotted as in B. (D) AKAP95 and Eg7 were immunoprecipitated from mitotic chromatin after solubilization with micrococcal nuclease and digestion with DNase I. Precipitates were immunoblotted as in B. (B–D) Control immunoprecipitations were carried out with preimmune rabbit IgGs (IgG). (E) Interphase nuclei (N) loaded with preimmune IgGs (−α-AKAP95), anti–AKAP95 antibodies (+α-AKAP95) or anti–HP1α antibodies (+α-HP1α) were allowed to condense in mitotic extract. After 2 h, chromatin masses (Ch) were sedimented and immunoblotted using anti–Eg7 antibodies.

Mentions: Several proteins have been shown to be required for mitotic chromosome condensation, including pEg7 (Cubizolles et al. 1998), a component of the 13S condensin complex (Hirano et al. 1997). To determine whether AKAP95 was part of the condensin complex or interacted with the complex, immunoprecipitations were carried out first from lysates of interphase or mitotic HeLa cells. Immunoblotting analysis of human Eg7 using a polyclonal antibody against the 15–carboxy-terminal amino acids of human Eg7 revealed that interphase and mitotic cells harbored similar levels of Eg7 (Fig. 10 A). Immunoprecipitation data showed that AKAP95 and Eg7 did not coprecipitate from interphase cell lysates, indicating that these proteins do not interact at this stage of the cell cycle (Fig. 10 B). In contrast, in mitotic cell lysates, AKAP95 and Eg7 coprecipitated regardless of the antibody used (Fig. 10 C), suggesting that AKAP95 and Eg7 interact at mitosis. Since AKAP95 was found to be associated with chromatin at mitosis (Fig. 1 C), we tested the possibility that the AKAP95–Eg7 interaction was mediated by DNA. To this end, mitotic chromatin was isolated, solubilized with micrococcal nuclease, and the DNA was digested with 400 μg/ml DNase I in the presence of protease inhibitors. Immunoprecipitation of AKAP95 or Eg7 from the DNase-treated fraction revealed that both proteins also coprecipitated (Fig. 10 D), suggesting that the mitotic AKAP95–Eg7 interaction is not mediated by DNA and is probably direct.


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)

Immunoblocking of AKAP95 inhibits the association of Eg7 to condensed chromatin in mitotic extract. (A) Immunoblot of interphase (I) and mitotic (M) HeLa cell lysates with antibodies against Eg7, a component of the human condensin complex. (B) AKAP95 and Eg7 were immunoprecipitated from interphase HeLa cell lysates and precipitates were immunoblotted with each precipitating antibody. (C) AKAP95 and Eg7 were immunoprecipitated from mitotic HeLa cell lysates and precipitates immunoblotted as in B. (D) AKAP95 and Eg7 were immunoprecipitated from mitotic chromatin after solubilization with micrococcal nuclease and digestion with DNase I. Precipitates were immunoblotted as in B. (B–D) Control immunoprecipitations were carried out with preimmune rabbit IgGs (IgG). (E) Interphase nuclei (N) loaded with preimmune IgGs (−α-AKAP95), anti–AKAP95 antibodies (+α-AKAP95) or anti–HP1α antibodies (+α-HP1α) were allowed to condense in mitotic extract. After 2 h, chromatin masses (Ch) were sedimented and immunoblotted using anti–Eg7 antibodies.
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Figure 10: Immunoblocking of AKAP95 inhibits the association of Eg7 to condensed chromatin in mitotic extract. (A) Immunoblot of interphase (I) and mitotic (M) HeLa cell lysates with antibodies against Eg7, a component of the human condensin complex. (B) AKAP95 and Eg7 were immunoprecipitated from interphase HeLa cell lysates and precipitates were immunoblotted with each precipitating antibody. (C) AKAP95 and Eg7 were immunoprecipitated from mitotic HeLa cell lysates and precipitates immunoblotted as in B. (D) AKAP95 and Eg7 were immunoprecipitated from mitotic chromatin after solubilization with micrococcal nuclease and digestion with DNase I. Precipitates were immunoblotted as in B. (B–D) Control immunoprecipitations were carried out with preimmune rabbit IgGs (IgG). (E) Interphase nuclei (N) loaded with preimmune IgGs (−α-AKAP95), anti–AKAP95 antibodies (+α-AKAP95) or anti–HP1α antibodies (+α-HP1α) were allowed to condense in mitotic extract. After 2 h, chromatin masses (Ch) were sedimented and immunoblotted using anti–Eg7 antibodies.
Mentions: Several proteins have been shown to be required for mitotic chromosome condensation, including pEg7 (Cubizolles et al. 1998), a component of the 13S condensin complex (Hirano et al. 1997). To determine whether AKAP95 was part of the condensin complex or interacted with the complex, immunoprecipitations were carried out first from lysates of interphase or mitotic HeLa cells. Immunoblotting analysis of human Eg7 using a polyclonal antibody against the 15–carboxy-terminal amino acids of human Eg7 revealed that interphase and mitotic cells harbored similar levels of Eg7 (Fig. 10 A). Immunoprecipitation data showed that AKAP95 and Eg7 did not coprecipitate from interphase cell lysates, indicating that these proteins do not interact at this stage of the cell cycle (Fig. 10 B). In contrast, in mitotic cell lysates, AKAP95 and Eg7 coprecipitated regardless of the antibody used (Fig. 10 C), suggesting that AKAP95 and Eg7 interact at mitosis. Since AKAP95 was found to be associated with chromatin at mitosis (Fig. 1 C), we tested the possibility that the AKAP95–Eg7 interaction was mediated by DNA. To this end, mitotic chromatin was isolated, solubilized with micrococcal nuclease, and the DNA was digested with 400 μg/ml DNase I in the presence of protease inhibitors. Immunoprecipitation of AKAP95 or Eg7 from the DNase-treated fraction revealed that both proteins also coprecipitated (Fig. 10 D), suggesting that the mitotic AKAP95–Eg7 interaction is not mediated by DNA and is probably direct.

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