Intrinsic disorder within AKAP79 fine-tunes anchored phosphatase activity toward substrates and drug sensitivity

Abstract

Scaffolding the calcium/calmodulin-dependent phosphatase 2B (PP2B, calcineurin) focuses and insulates termination of local second messenger responses. Conformational flexibility in regions of intrinsic disorder within A-kinase anchoring protein 79 (AKAP79) delineates PP2B access to phosphoproteins. Structural analysis by negative-stain electron microscopy (EM) reveals an ensemble of dormant AKAP79-PP2B configurations varying in particle length from 160 to 240 Å. A short-linear interaction motif between residues 337–343 of AKAP79 is the sole PP2B-anchoring determinant sustaining these diverse topologies. Activation with Ca2+/calmodulin engages additional interactive surfaces and condenses these conformational variants into a uniform population with mean length 178 ± 17 Å. This includes a Leu-Lys-Ile-Pro sequence (residues 125–128 of AKAP79) that occupies a binding pocket on PP2B utilized by the immunosuppressive drug cyclosporin. Live-cell imaging with fluorescent activity-sensors infers that this region fine-tunes calcium responsiveness and drug sensitivity of the anchored phosphatase.Signaling molecules such as the hormone epinephrine (also known as adrenaline) activate a range of responses inside cells. The responses often involve proteins being chemically modified to change how active they are, which in turn controls specific processes happening inside the cell. One type of modification involves certain enzymes adding or removing molecules known as phosphate groups from specific proteins. For example, an enzyme called PP2B (also known as calcineurin) is able to remove phosphate groups from a variety of proteins.PP2B plays crucial roles in many different processes in animals including immune responses, nerve cell signaling and heart activity, and is the target of several medicinal drugs that suppress the immune system. Since PP2B plays so many roles in the body, these drugs often have unintended side effects. Therefore, studying how the body regulates this enzyme may help us to understand what causes these side effects.Previous studies have shown that PP2B is activated by calcium ions, which can act as signals in many different situations inside cells. A protein called AKAP79 anchors PP2B to specific locations in the cell so that it only operates where it is needed. Some evidence suggests that calcium ions affect how AKAP79 and PP2B interact, but it is not known how this works. Nygren et al. investigated how the PP2B enzyme and AKAP79 protein interact inside human cells and in cell-free systems.The experiments showed that short regions within the AKAP79 protein are responsible for binding to PP2B. These regions and the flexible structure of the entire AKAP79 protein work together to fine-tune how PP2B responds to calcium ions. In the presence of higher levels of calcium ions, another ‘auxiliary’ region of AKAP79 also binds to PP2B. This auxiliary region binds to a site on the enzyme where an immunosuppressive drug called cyclosporine can also bind. This suggests that AKAP79 binding to PP2B may affect the sensitivity of the PP2B enzyme to cyclosporine.This study demonstrates that the activity of PP2B can be precisely controlled by interactions with proteins such as AKAP79. Further work on these interactions may help develop more effective drugs that cause fewer side effects in patients.