Conformational switching on AKAP7 modulates the phosphorylation dynamics of anchored PKC

Abstract

The spatiotemporal fidelity of signal transduction is maintained in large part by scaffolding proteins. In particular, A‐Kinase Anchoring Proteins (AKAPs) accelerate and amplify the phosphorylation of substrate proteins by anchored kinases. However, the mechanisms underlying these phenomena remain unclear. Our present study endeavors to clarify this mechanism utilizing a combination of mathematical modeling, biochemistry, and FRET microscopy. We propose a model wherein enzymes on a scaffold undergo a “conformational switch” into an active intermediate. This model predicts anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition in comparison to those occuring in solution. We are exploring a novel interaction between AKAP7α and Protein Kinase C (PKC) as a model system to validate these predictions. Our preliminary data comparing the FRET responses of a genetically encoded PKC activity reporter (CKAR) to an AKAP7α‐CKAR fusion protein reveal that both the speed and strength of the CKAR substrate phosphorylation is enhanced when it is tethered to AKAP7α. Additionally, PKC bound to AKAP7α appears to be less susceptible to several classes of PKC inhibitors, including the ATP competitive inhibitor Gö6976 and the substrate inhibitor PKC 20–28. Collectively, our findings provide strong theoretical and molecular evidence that more narrowly defines the anchoring hypothesis.