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Regulation of anchoring of the RII regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RII: implications for chromosome dynamics at mitosis

¹ Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway
² Ruhr Universität Bochum, Institut für Physiologische Chemie, MA2 Nord Raum 40, 44780 Bochum, Germany

View larger version (61K): [in a new window]   Fig. 1. Subcellular distribution of AKAP95 and RII in Reh, Reh-RII and Reh-RII(T54E) cells. (A) Assessment of AKAP95 and RII in Reh, Reh-RII and Reh-RII(T54E) cells. Lysates from interphase and mitotic cells (106 cells per lane) were analyzed by immunoblotting using mAbs against human AKAP95 (mAb47) and human RII. (B,C) Immunofluorescence analysis of interphase (B) and mitotic (C) cells using affinity-purified anti-AKAP95 polyclonal antibodies (green) and anti-RII mAbs (red). DNA was counterstained with Hoechst 33342 (blue). Bars, 10 µm.

View larger version (36K): [in a new window]   Fig. 2. Wild-type human RII, but not RII(T54E), co-fractionates with chromatin at mitosis. (A) Mitotic Reh-RII cells (upper panels) and mitotic Reh-RII(T54E) cells (lower panels) were homogenized and lysates centrifuged at 15,000 g. The sedimented material (P15) was extracted with 1% Triton X-100 and the Triton-X-100-insoluble material was treated with MNase and sedimented to produce MNase-soluble (MNase (S)) and MNase-insoluble (MNase (P)) fractions. The 15,000 g supernatants from the first centrifugation (S15) were fractionated at 200,000 g into soluble (S200) and particulate (P200) fractions. Fractions were immunoblotted using anti-AKAP95 and anti-RII mAbs. (B) Mitotic rat PC12 cells were fractionated into P15, MNase-soluble (S) and MNase-insoluble (P) fractions, and each fraction was immunoblotted as in (A) and using anti-RIIß mAbs.

View larger version (27K): [in a new window]   Fig. 3. Association of AKAP95 and RII at mitosis. (A) AKAP95 and RII were immunoprecipitated (IP) from interphase (I) or metaphase (M) lysates of Reh-RII and Reh-RII(T54E) cells, and immunoprecipitates were immunoblotted using anti-AKAP95 and anti-RII mAbs (Blot). (B) MNase-soluble chromatin (Chr) and a cytosolic fraction (Cyt) were prepared from mitotic Reh-RII or Reh-RII(T54E) cells. AKAP95 was immunoprecipitated and the precipitates immunoblotted using anti-RII mAbs.

View larger version (34K): [in a new window]   Fig. 4. Alkaline phosphatase disrupts the mitotic AKAP95-RII interaction. (A) Anti-RII (first panel) and anti-AKAP95 immunoprecipitates (second panel) from interphase (I) and mitotic (M) Reh-RII and Reh-RII(T54E) cells were immunoblotted using anti-pT and anti-pS antibodies. (B) Soluble chromatin was prepared from mitotic Reh-RII cells and treated with 100 U ml-1 APase (APase+) or 100 U ml-1 APase plus 20 mM sodium vanadate (APase-). AKAP95 or RII were immunoprecipitated (IP) and immune precipitates immunoblotted using anti-AKAP95 and anti-RII mAbs. (C) Duplicates of blots were immunoblotted using anti-pT and anti-pS antibodies.

View larger version (21K): [in a new window]   Fig. 5. Recruitment of RII onto Reh chromatin in mitotic extract derived from Reh-RII, but not Reh-RII(T54E), cells. Purified Reh nuclei were incubated for 2 hours in extract derived from mitotic Reh, Reh-RII(T54E) or Reh-RII cells to elicit chromatin condensation. (A) Localization of AKAP95 (red) and RII (green) was assessed by immunofluorescence analysis of condensed chromatin. Bar, 10 µm. (B) Condensed chromatin masses were recovered by sedimentation through sucrose and proteins immunoblotted using anti-AKAP95 and anti-RII mAbs. (C) AKAP95 and RII were immunoprecipitated from each condensed chromatin fraction after solubilization with MNase, and immune precipitates were immunoblotted using anti-pT antibodies.

View larger version (29K): [in a new window]   Fig. 6. Disruption of AKAP-RII anchoring induces reversible premature chromatin decondensation. (A) Reh-RII chromatin condensed in mitotic Reh-RII extract was recovered and incubated in fresh mitotic Reh-RII extract containing 500 nM Ht31, 500 nM control Ht31-P peptide or no peptide. Percentage of PCD was monitored over 3 hours after DNA staining. (B) Chromatin (P) and reaction supernatants (S) before (Input) and after a 3-hour incubation in extract containing Ht31 or Ht31-P as in (A) were analyzed by immunoblotting using anti-AKAP95 and anti-RII mAbs. (C) Prematurely decondensed Reh-RII chromatin was recovered from Ht31-containing extract and resuspended in fresh mitotic Reh extract containing no peptide or increasing concentrations of recombinant wild-type RII or indicated mutants. Proportions of PCD were monitored after 1.5 hours by DNA staining. (D) Chromatin masses obtained at the end of incubation with wild-type RII or RII mutants were isolated and immunoblotted using anti-AKAP95 and anti-RII antibodies. (E) Reh nuclei were condensed for 2 hours in mitotic Reh extract. Condensed chromatin was recovered and incubated (T=0 hours) in mitotic extract of Reh, Reh-RII or Reh-RII(T54E) cells. The percentage of PCD was evaluated over 2 hours after DNA staining. (F) Reh nuclei were condensed for 2 hours in mitotic Reh extract as in (E). Condensed chromatin was recovered and incubated (T=0 hours) in Reh-RII mitotic extract immunodepleted for RII, or in Reh extract containing 11 nM of either wild-type RII or RII(T54E). The percentage of PCD was evaluated over 2 hours after DNA staining.

View larger version (32K): [in a new window]   Fig. 7. Nuclear reassembly in vitro is accompanied by dissociation of RII from chromatin-bound AKAP95 and threonine dephosphorylation of RII. Mitotic Reh-RII cells were lysed by Dounce homogenization and nuclei were allowed to reform by incubating the lysate at 30°C. (A) At the indicated time points, solubilization of RII and reassembly of nuclear membranes were monitored by immunofluorescence using anti-RII mAbs (green) and affinity-purified antibodies against LBR, an integral protein of the inner nuclear membrane (red); merged images are shown (upper panels). DNA was labeled with Hoechst 33342 (lower panels). Bar, 10 µm. (B) Input (0 minutes) and decondensed chromatin fractions (75 minutes) were sedimented and pellets (P) and supernatants (S) immunoblotted using anti-AKAP95 and anti-RII mAbs. (C) Entire nuclear reassembly reactions were homogenized at the indicated time points, RII was immunoprecipitated and precipitates were immunoblotted using anti-pT antibodies. RII was also immunoprecipitated after a 75-minute control incubation of chromatin in mitotic lysate without an ATP-generating system (M 75 min).

View larger version (19K): [in a new window]   Fig. 8. Model for how reversible AKAP95-PKA interaction (mediated by RII phosphorylation) controls chromatin structure during mitosis. During interphase, AKAP95 and PKA-RII localize to distinct compartments, separated by the nuclear envelope. At mitosis entry, AKAP95 associates with condensing chromatin. RII is phosphorylated by the CDK1-cyclin-B complex. RII phosphorylation turns on a molecular switch promoting RII anchoring to AKAP95 and maintenance of condensed chromosomes during mitosis. (Although anchoring of RII to AKAP95 has been demonstrated, anchoring of the catalytic subunit of PKA is only suggested.) Throughout mitosis, anchoring of phosphorylated RII to chromatin-bound AKAP95 is required to prevent PCD. At mitosis exit, dephosphorylation of RII by a threonine phosphatase induces RII dissociation from chromatin-bound AKAP95. This relieves inhibition of chromatin decondensation, allowing chromosome decondensation as the nuclear envelope reassembles.

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