Analysis of AKAP7? 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 AKAP7? self-associates. Self-association appears to be an isoform specific phenomenon, as AKAP7? did not associate with itself or with AKAP7?. However, AKAP7? did associate with AKAP7?, suggesting the long isoforms of the AKAP can form heterodimers. Surface plasmon resonance found that the AKAP7? 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 AKAP7? interacts with itself through multiple regions. Photon counting histogram analysis (PCH) of AKAP7?-EGFP expressed in HEK-293 cells confirmed that AKAP7?-EGFP self-associates in a cellular context. Lastly, computational modeling of PKA dynamics within AKAP7? complexes suggests that oligomerization may augment phosphorylation of scaffolded PKA substrates. In conclusion, our study reveals that AKAP7? 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.

SUBMITTER: Singh A 

PROVIDER: S-EPMC4568377 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature