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J Cell Biol
1999 Dec 13;1476:1167-80. doi: 10.1083/jcb.147.6.1167.
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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
.
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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.
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 antiâAKAP95 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 antiâAKAP95 antibody. (C) Interphase and mitotic HeLa cells (107 and 5 à 107, respectively) were fractionated into nuclei or chromatin, cytosol, and cytoplasmic membranes, and proteins of each fraction were immunoblotted using antiâAKAP95 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 saltâextracted nuclear matrices, proteins were immunoblotted using antiâAKAP95 antibodies, and duplicate blots analyzed by densitometry.
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 2â5 pg affinity-purified polyclonal antiâAKAP95 antibodies or antiâAKAP95 antibodies together with 250 pg competitor GST-AKAP95Î1-386 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 30â40 cells injected per treatment. Arrow points to a noninjected cell that did not undergo PCD. Bars, 10 μm.
Figure 8. AKAP95âRIIα 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 antiâAKAP95 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 antiâAKAP95 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 antiâAKAP95 and antiâRIIα antibodies.
Figure 9. Disruption of AKAP95âRII 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, â¼1 ng C, or â¼1 ng C together with 2â5 pg antiâAKAP95 antibodies. Cells remained in nocodazole after injection and were examined as in Fig. 7. Percentage of PCD was calculated from 30â40 injected cells. Bars, 10 μm.
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. (BâD) Control immunoprecipitations were carried out with preimmune rabbit IgGs (IgG). (E) Interphase nuclei (N) loaded with preimmune IgGs (âα-AKAP95), antiâAKAP95 antibodies (+α-AKAP95) or antiâHP1α antibodies (+α-HP1α) were allowed to condense in mitotic extract. After 2 h, chromatin masses (Ch) were sedimented and immunoblotted using antiâEg7 antibodies.
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