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

CDK1 phosphorylates the A-kinase regulatory subunit RIIα on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIα. Using an RIIα-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIα or an RIIα(T54E) mutant, we show that RIIα associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIα on T54. During interphase, both RIIα and RIIα(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIα, but not RIIα(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIα. Disrupting AKAP95-RIIα anchoring or depleting RIIα from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIα is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondensation in vitro, which can be rescued by addition of wild-type RIIα or an RIIα(T54D) mutant, but not RIIα(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIα constitutes a molecular switch controlling anchoring of RIIα to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.