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

CDK1 phosphorylates the A-kinase regulatory subunit RII alpha on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localizatio n of RII alpha. Using an RII alpha -deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RII alpha. or an RI I alpha (T54E) mutant, we show that RII alpha associates with chromatin-bou nd A-kinase anchoring protein AKAP95 at mitosis and that this interaction i nvolves phosphorylation of RII alpha: on T54. During interphase, both Rita and RII alpha (T54E) exhibit a centrosome-Golgi localization, whereas AKAP9 5 is intranuclear. At mitosis and in a mitotic extract, most RII alpha, but not RII alpha (T54E), cofractionates with chromatin, onto which it associa tes with AKAP95. This correlates with T54 phosphorylation of RII alpha. Dis rupting AKAP95-RII alpha anchoring or depleting RII alpha from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstit ution assay that mimics mitotic nuclear reformation, RII alpha is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondens ation in vitro, which can be rescued by addition of wild-type RII alpha or an RII alpha (T54D) mutant, but not RII alpha (T54E, A, L or V) mutants. Ou r results suggest that CDK1-mediated T54 phosphorylation of RII alpha const itutes a molecular switch controlling anchoring of RII alpha to chromatin-b ound AKAP95, where the PKA-AKAP95 complex participates in remodeling chroma tin during mitosis.