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Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication
Turid Eide1, Kristin A Taskén, Cathrine Carlson
1Department of Medical Biochemistry, University of Oslo, P. O. Box 1112 Blindern, 0317 Oslo, Norway.
Abstract:
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.
Insights
Protein kinase A (PKA)-anchoring protein AKAP95 acts as a scaffold for minichromosome maintenance 2 (MCM2) protein, crucial for DNA replication initiation and elongation in the nucleus.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear matrix protein AKAP95 anchors Protein Kinase A (PKA).
- AKAP95 localizes to the nucleus during interphase.
- Minichromosome maintenance (MCM) 2 is a key component of the pre-replication complex.
Purpose of the Study:
- To investigate the interaction between AKAP95 and MCM2.
- To elucidate the role of AKAP95 in DNA replication.
- To determine the functional significance of the AKAP95-MCM2 interaction.
Main Methods:
- Yeast two-hybrid screening to identify AKAP95 interaction partners.
- Glutathione S-transferase precipitation and immunoprecipitation to confirm protein interactions.
- In vitro replication assays using HeLa cell nuclei and peptide inhibitors.
- Depletion and re-introduction of AKAP95 to assess its role in replication.
Main Results:
- AKAP95 interacts with MCM2, specifically mapping to residues 1-195 of AKAP95.
- Disrupting the AKAP95-MCM2 interaction abolishes DNA replication initiation and elongation.
- AKAP95 depletion inhibits replication, which can be restored by recombinant AKAP95.
- PKA inhibition affects replication initiation but not elongation, suggesting distinct roles.
Conclusions:
- AKAP95 serves as a crucial scaffold protein for MCM2 in the nucleus.
- AKAP95 plays a vital role in regulating DNA replication initiation and elongation.
- The AKAP95-MCM2 interaction is essential for efficient DNA replication.