Analysis of AKAP7γ Dimerization.

Arpita Singh,Cathrine R Carlson,Marc Rigatti,Kimberly L Dodge-Kafka,Ion I Moraru,Andrew V Le

doi:10.1155/2015/371626

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.

Highlights

Phosphorylation of target proteins is a key mechanism utilized by the cell to induce changes in physiology and function
We have previously found that the long isoform AKAP7γ associates with Protein Kinase C (PKC), PP1, the PP1 inhibitor-1 (I-1), and phospholamban [16, 21,22,23] and others have shown that it interacts with both PDE4D3 and PDE3A [20, 24]
In order to understand the molecular architecture of the AKAP7γ signaling complex and determine if the A-Kinase anchoring protein surface plasmon resonance analysis (SPR) (AKAP) can be the basis for higher order structures, we first looked to see if purified, recombinant AKAP7γ could pull down an AKAP7γ complex when transiently expressed in cells

Summary

Introduction

Phosphorylation of target proteins is a key mechanism utilized by the cell to induce changes in physiology and function. Recent work has suggested that the subcellular localization of signaling enzymes to discrete locations in the cell confers spatiotemporal control over phosphorylation events [1]. This is facilitated by scaffolding proteins, which tether the kinase to the same signaling complex as its substrates, allowing for increased speed and amplitude of phosphorylation events [2]. Oligomerization of β-arrestin sequesters the scaffold in the cytoplasm, inhibiting its nuclear actions [7]. These examples demonstrate that oligomerization greatly impacts the function of the scaffold

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Conclusion