Therapeutic strategies for anchored kinases and phosphatases: exploiting short linear motifs and intrinsic disorder

Abstract

Phosphorylation events that occur in response to the second messenger cAMP are controlled spatially and temporally by protein kinase A (PKA) interacting with A-kinase anchoring proteins (AKAPs). Recent advances in understanding the structural basis for this interaction have reinforced the hypothesis that AKAPs create spatially constrained signaling microdomains. This has led to the realization that the PKA/AKAP interface is a potential drug target for modulating a plethora of cell-signaling events. Pharmacological disruption of kinase-AKAP interactions has previously been explored for disease treatment and remains an interesting area of research. However, disrupting or enhancing the association of phosphatases with AKAPs is a therapeutic concept of equal promise, particularly since they oppose the actions of many anchored kinases. Accordingly, numerous AKAPs bind phosphatases such as protein phosphatase 1 (PP1), calcineurin (PP2B), and PP2A. These multimodal signaling hubs are equally able to control the addition of phosphate groups onto target substrates, as well as the removal of these phosphate groups. In this review, we describe recent advances in structural analysis of kinase and phosphatase interactions with AKAPs, and suggest future possibilities for targeting these interactions for therapeutic benefit.

Keywords: A-kinase anchoring protein (AKAP); cAMP signaling; calcineurin; intrinsic disorder; protein kinase A (PKA); protein phosphatase 2B (PP2B); short linear interaction motifs (SLiMs).

FIGURE 1

Structural basis for protein kinase A (PKA) holoenzyme formation and anchoring. (A) Crystal structure of the synthetic A-kinase anchoring protein (AKAP) helix AKAP’s (orange) in complex with the RIIα docking/dimerization (D/D) domain, residues 1–44 (blue). The AKAP amphipathic helix binds to a hydrophobic groove created by the antiparallel X-type helix bundle of the RII D/D domain. PDB ID: 2IZX. (B) Left: RIIβ cAMP-binding cassettes (blue) in complex with cAMP (red). Right: RIIα cAMP binding cassettes in complex with PKA catalytic subunit (green). With cAMP bound at each of two sites, RII releases inhibition of the catalytic subunit. When cAMP is not present, RII presents an inhibitory sequence to the active site, preventing phosphorylation of PKA substrates. PDB IDs: 1CX4, 2WVS. (C) A pseudo-atomic model of the PKA holoenzyme in complex with AKAP18γ derived from low-resolution EM data. This illustrates that the PKA holoenzyme has a constrained range of flexibility (∼300 Å) provided by AKAPs, allowing the catalytic subunits to be poised near potential substrates. PDB IDs: 3J4Q, 3J4R. Models were prepared using PyMol (Schrödinger).

FIGURE 2

Structural basis for phosphatase regulation and anchoring. (A) Left: PP1 catalytic subunit (gray) in complex with RVxF and auxiliary anchoring motifs from protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS; orange). Right: PP2B (gray) in complex with PIAIIIT sequence from AKAP79 (orange). Comparison reveals that similar surfaces are used for anchoring, and that multiple motifs can simultaneously interact with varied portions of the molecule. PDB IDs: 4MOY, 3LL8. (B) Left: PP2B in complex with cyclosporin (red)/cyclophilin (yellow) complex. Right: PP2B in complex with a viral peptide A238L, containing a PxIxIT motif, as well as an LxVP motif (red). Cyclosporin and LxVP peptides bind to overlapping surfaces on PP2B, formed by both the catalytic and regulatory subunits of PP2B. This surface does not occlude the active site of the phosphatase, yet immunosuppressants are able to allosterically inhibit PP2B activity toward substrates. PDB IDs: 1MF8, 4F0Z. Models were prepared using PyMol (Schrödinger).