title: Analysis of AKAP7γ Dimerization.
abstract: A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that contribute to spatiotemporal regulation of PKA-mediated phosphorylation events. In particular, AKAP7 is a family of alternatively spliced proteins that participates in cardiac calcium dynamics. Here, we demonstrate via pull-down from transfected cells and by direct protein-protein association that AKAP7γ self-associates. Self-association appears to be an isoform specific phenomenon, as AKAP7α did not associate with itself or with AKAP7γ. However, AKAP7γ did associate with AKAP7δ, suggesting the long isoforms of the AKAP can form heterodimers. Surface plasmon resonance found that the AKAP7γ self-association occurs via two high affinity binding sites with K D values in the low nanomolar range. Mapping of the binding sites by peptide array reveals that AKAP7γ interacts with itself through multiple regions. Photon counting histogram analysis (PCH) of AKAP7γ-EGFP expressed in HEK-293 cells confirmed that AKAP7γ-EGFP self-associates in a cellular context. Lastly, computational modeling of PKA dynamics within AKAP7γ complexes suggests that oligomerization may augment phosphorylation of scaffolded PKA substrates. In conclusion, our study reveals that AKAP7γ forms both homo- and heterodimers with the long isoforms of the AKAP and that this phenomenon could be an important step in mediating effective substrate phosphorylation in cellular microdomains.
authors.affiliation: Pat and Jim Calhoun Center for Cardiology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Arpita
authors.initials: A
authors.lastname: Singh
authors.affiliation: Pat and Jim Calhoun Center for Cardiology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA ; The Richard D. Berlin Center for Cell Analysis & Modeling, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Rigatti
authors.affiliation: Pat and Jim Calhoun Center for Cardiology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Le
authors.affiliation: Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway ; KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway.
authors.forename: Cathrine R
authors.initials: CR
authors.lastname: Carlson
authors.affiliation: The Richard D. Berlin Center for Cell Analysis & Modeling, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Ion I
authors.initials: II
authors.lastname: Moraru
authors.affiliation: Pat and Jim Calhoun Center for Cardiology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Dodge-Kafka