Regulation of anchoring of the RIIalpha regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIalpha: implications for chromosome dynamics at mitosis.
CDK1 phosphorylates the A-kinase regulatory subunit RIIalpha on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIalpha. Using an RIIalpha-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIalpha or an RIIalpha(T54E) mutant, we show that RIIalpha associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIalpha on T54. During interphase, both RIIalpha and RIIalpha(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIalpha, but not RIIalpha(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIalpha. Disrupting AKAP95-RIIalpha anchoring or depleting RIIalpha from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIalpha 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 RIIalpha or an RIIalpha(T54D) mutant, but not RIIalpha(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIalpha constitutes a molecular switch controlling anchoring of RIIalpha to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.
Landsverk
H B
HB
Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway.
Carlson
C R
CR
Steen
R L
RL
Vossebein
L
L
Herberg
F W
FW
Taskén
K
K
Collas
P
P
D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism*; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation / Q000502:physiology; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*
C490117:AKAP8L protein, human; D002843:Chromatin; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C517819:PRKAR2A protein, human; D013912:Threonine; D017868:Cyclic AMP-Dependent Protein Kinases