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.

Mechanisms for localising calcineurin and CaMKII in dendritic spines

“…1b. Calcineurin interacts with the AKAP79 PIAIIITD motif in the low μM range [39,41], consistent with high occupation of the site in dendritic spines given the high concentration of calcineurin in the brain [4].…”

“…In addition, gas phase measurements [40] indicate that calcineurin can bind in a ratio of 2 calcineurin: 1 AKAP79. Conversely, interaction assays in solution [39,41], fluorescent imaging in cultured cells [39], and imaging by electron microscopy [41] support a binding ratio of 1 AKAP79: 1 calcineurin, with calcineurin binding to a single side of the PIAIIITD β-strand, as shown in Fig. 1b.…”

“…Calcineurin-interacting proteins may contain either or both of the two motif types, and there is no clear rule dictating spacing when both motifs are present [45]. AKAP79 is an example of a bipartite calcineurin-interacting protein [40], with the LxVP-type sequence 'LKIP' in its N-terminus mediating Ca 2+ /CaM-dependent interactions with calcineurin [41]. Single particle analysis by electron microscopy shows that Ca 2+ /CaM triggers a contraction in the AKAP79-calcineurin complex brought about by association of the AKAP79 LKIP sequence [41] with activated calcineurin.…”

“…AKAP79 is an example of a bipartite calcineurin-interacting protein [40], with the LxVP-type sequence 'LKIP' in its N-terminus mediating Ca 2+ /CaM-dependent interactions with calcineurin [41]. Single particle analysis by electron microscopy shows that Ca 2+ /CaM triggers a contraction in the AKAP79-calcineurin complex brought about by association of the AKAP79 LKIP sequence [41] with activated calcineurin. The functional effects of removing LKIP from AKAP79 are subtle in comparison to removing the PIAIIITD calcineurin anchoring site [41].…”

Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins

“…Negative-stain electron microscopy and 3D reconstructions show that intact hetero-pentameric AKAP-RII 2 -C 2 assemblies adopt a range of flexible tripartite configurations (21,22). This is because intrinsically disordered regions between the D/ D domains and cAMP-binding sites on each regulatory subunit permit a 150-to 200-Å radius of motion to the associated catalytic subunits (21,23). Such flexibility within anchored PKA holoenzyme complexes enables precise orientation of the catalytic subunit toward substrates (11,24).…”