PKA-Type I Selective Constrained Peptide Disruptors of AKAP Complexes

Abstract

A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.

Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα

“…Concomitant with these shifts in the activation constant, we observed a positive correlation between activation of the kinase and helicity (Figure S7). We propose that stabilization of this helix by chemical means, as has been demonstrated with AKAP disruptors, may pose an additional avenue for further development of these activators (Kennedy and Scott, 2015; Wang et al, 2015). …”

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry

“…It will be interesting to translate our findings to the cellular environment, where subcellular PKAc complexes with differential affinity for PKI could exist, with inhibitor affinity regulated as a function of PKAc phosphorylation or heterodimerization. In this regard, the analysis of PKI binding to nonphosphorylated, myristoylated or mutated PKAc, or PKAc complexes disrupted by cell-permeable AKAP ligands [76], will be of particular interest.…”

Targeting kinase signaling pathways with constrained peptide scaffolds

“…AKAPs are highly diverse in function and localization, but share the common defining feature of anchoring PKA in an isoform-specific manner via interactions with the regulatory subunits of PKA (PKA-R) (Kennedy & Scott, 2015). Although numerous peptide inhibitors were previously designed to target the AKAP-PKA interface as a means to regulate PKA in a spatial and temporal manner (reviewed in (Kennedy & Scott, 2015; Troger, Moutty, Skroblin, & Klussmann, 2012), cell-permeant “stapled” alpha-helical peptides were more recently designed to disrupt the same PPI interface (Wang, et al, 2014; Wang, et al, 2015). These stapled AKAP inhibitors have the added advantage of being both cell permeant and constrained in a pre-binding state.…”

“…However, a limitation of targeting this particular PPI is that current AKAP inhibitors act as universal disruptors by blocking PKA anchoring in an isoform-specific manner, i.e. all RI- or all RII-selective AKAPs (Kennedy & Scott, 2015; Troger, et al, 2012; Wang, et al, 2015), and therefore no cell-permeant inhibitors exist which target individual AKAPs. Since most cells express 10–15 AKAPs simultaneously (Lester, Coghlan, Nauert, & Scott, 1996), selective disruption of individual AKAPs will be ideal for investigating an AKAP of interest.…”