Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication.
Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.
Department of Medical Biochemistry, University of Oslo, P. O. Box 1112 Blindern, 0317 Oslo, Norway.
Turid
T
Eide
Kristin A
KA
Taskén
Cathrine
C
Carlson
Gareth
G
Williams
Tore
T
Jahnsen
Kjetil
K
Taskén
Philippe
P
Collas
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C490117:AKAP8L protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D064111:Minichromosome Maintenance Complex Component 2