Langevin Dynamics Simulation of AKAP-PKA Complex: Re-Envisioning the Local Concentration Mechanism for Directing PKA Phosphorylation

Abstract The second messenger cAMP and its effector cAMP-dependent protein kinase A (PKA) constitute a ubiquitous cell signaling system. In its inactive state PKA is composed of two regulatory subunits that dimerize, and two catalytic subunits that are inhibited by the regulatory subunits. Activation of the catalytic subunits occurs upon binding of two molecules of cAMP to each regulatory subunit. Although many receptor types existing within the same cell may use this signaling system, compartmentation of signaling is thought to occur due to A-Kinase Anchoring Proteins (AKAPs), which act to co-localize PKA with specific substrates. However, the molecular mechanism allowing AKAPs to direct PKA phosphorylation to a particular substrate remained elusive, as prior evidence suggested that the catalytic subunit, which is highly diffusible, is released after cAMP binding to the regulatory subunit. Recent evidence from Smith et al. suggests that in the cell, the catalytic subunit may in fact not be released from the AKAP complex 1, 2. They further demonstrated that alterations in the structure of the PKA regulatory subunit tether affect substrate phosphorylation. We use a novel computational software based on Langevin dynamics, SpringSaLaD, to simulate the AKAP-PKA complex in order to determine a molecular mechanism for the changes in phosphorylation seen with alteration in tether length and flexibility, and to demonstrate whether or not AKAPs can effectively direct PKA phosphorylation to a particular substrate upon release of the catalytic subunit from the complex. We find that short and flexible tethers contribute to a decrease in the average characteristic time of binding, allowing the catalytic subunit to spend more time in a bound state with the substrate, which yields faster characteristic times of phosphorylation. We further demonstrate that release of the catalytic subunit from the AKAP complex abrogates the effect of tethering, with characteristic times of phosphorylation similar to non-AKAP bound PKA. The data demonstrates that AKAPs likely do not release the catalytic subunit in directing PKA phosphorylation to AKAP bound substrates. In combination with the changes in characteristic time of phosphorylation which are driven by tether structure, this work indicates that the purpose of AKAPs may be to increase the efficiency of phosphorylation of particular AKAP substrates.