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Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation.

Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T - Elife (2013)

Bottom Line: Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits.Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates.We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, United States.

ABSTRACT
Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.

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Flexibility within RIIα constrains the configuration of the anchored kinase assembly.(A) Amino acid sequence alignment of the linker region in RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains. This region shows low sequence homology and is likely to be structurally disordered. (B) Schematic representations of modified AKAP18γ–PKAholo complexes with AKAP18γ depicted in yellow, RIIα in green and PKAc in blue. (C) Biochemical analysis of the purified AKAP18γ–RIIα-PKAc complexes assembled with the wild-type RIIα subunit, the RIIα Δ44–86 mutant where the linker region was deleted, and the RIIα ZeChimera mutant where the mouse RIIα linker region was replaced with the corresponding and extended sequence from zebrafish. Top panel shows SDS-PAGE and Coomassie blue staining of the protein components. The next three panels show western blotting for RIIα, PKAc subunit and AKAP18γ, respectively. (D) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα Δ44–86 construct. (E) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα ZeChimera construct. Insets in (D and E) show enlarged views of individual particles outlined in gold for clarity. (F) Projection averages of the AKAP18γ–PKAholo complexes assembled with a truncated RIIα Δ44–86 construct using ISAC (Yang et al., 2012). (G) Projection averages of AKAP18γ–PKAholo complex assembled an RIIα ZeChimera construct. Scale bars in (F) and (G) represent 25 nm. (H) Statistical analysis of particle radius in angstroms (Å) for each AKAP18γ–PKAholo complexes. Box plot displays second and third quartile values, tails corresponding to minimum and maximum distances, (**) indicates p<0.01; (****) indicates p<0.0001.DOI:http://dx.doi.org/10.7554/eLife.01319.008
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fig3: Flexibility within RIIα constrains the configuration of the anchored kinase assembly.(A) Amino acid sequence alignment of the linker region in RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains. This region shows low sequence homology and is likely to be structurally disordered. (B) Schematic representations of modified AKAP18γ–PKAholo complexes with AKAP18γ depicted in yellow, RIIα in green and PKAc in blue. (C) Biochemical analysis of the purified AKAP18γ–RIIα-PKAc complexes assembled with the wild-type RIIα subunit, the RIIα Δ44–86 mutant where the linker region was deleted, and the RIIα ZeChimera mutant where the mouse RIIα linker region was replaced with the corresponding and extended sequence from zebrafish. Top panel shows SDS-PAGE and Coomassie blue staining of the protein components. The next three panels show western blotting for RIIα, PKAc subunit and AKAP18γ, respectively. (D) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα Δ44–86 construct. (E) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα ZeChimera construct. Insets in (D and E) show enlarged views of individual particles outlined in gold for clarity. (F) Projection averages of the AKAP18γ–PKAholo complexes assembled with a truncated RIIα Δ44–86 construct using ISAC (Yang et al., 2012). (G) Projection averages of AKAP18γ–PKAholo complex assembled an RIIα ZeChimera construct. Scale bars in (F) and (G) represent 25 nm. (H) Statistical analysis of particle radius in angstroms (Å) for each AKAP18γ–PKAholo complexes. Box plot displays second and third quartile values, tails corresponding to minimum and maximum distances, (**) indicates p<0.01; (****) indicates p<0.0001.DOI:http://dx.doi.org/10.7554/eLife.01319.008

Mentions: Our model of the anchored PKA complex implies that an intrinsically disordered flexible linker region within RIIα supports the array of conformations that were observed in the raw micrographs and the projection averages (Figures 1 and 2C). To quantitatively assess the distribution of conformations assumed by this complex, we measured the end-to-end distance between the two large peripheral densities of 223 individual particles (Figure 2D). This population of structures followed a Gaussian distribution (Figure 2D, green trace) with a mean particle length of 275 ± 65 Å (n = 223). We propose that conformational plasticity observed in these analyses is facilitated by this intrinsically disordered region between residues 44 and 90 of RIIα, a linker that connects the AKAP docking site (D/D domain) to the cAMP-responsive transduction domains. This notion is further substantiated by a primary sequence analysis of RIIα orthologs, showing that the linker regions are of similar length but exhibit low amino acid identity (Figure 3A).10.7554/eLife.01319.008Figure 3.Flexibility within RIIα constrains the configuration of the anchored kinase assembly.


Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation.

Smith FD, Reichow SL, Esseltine JL, Shi D, Langeberg LK, Scott JD, Gonen T - Elife (2013)

Flexibility within RIIα constrains the configuration of the anchored kinase assembly.(A) Amino acid sequence alignment of the linker region in RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains. This region shows low sequence homology and is likely to be structurally disordered. (B) Schematic representations of modified AKAP18γ–PKAholo complexes with AKAP18γ depicted in yellow, RIIα in green and PKAc in blue. (C) Biochemical analysis of the purified AKAP18γ–RIIα-PKAc complexes assembled with the wild-type RIIα subunit, the RIIα Δ44–86 mutant where the linker region was deleted, and the RIIα ZeChimera mutant where the mouse RIIα linker region was replaced with the corresponding and extended sequence from zebrafish. Top panel shows SDS-PAGE and Coomassie blue staining of the protein components. The next three panels show western blotting for RIIα, PKAc subunit and AKAP18γ, respectively. (D) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα Δ44–86 construct. (E) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα ZeChimera construct. Insets in (D and E) show enlarged views of individual particles outlined in gold for clarity. (F) Projection averages of the AKAP18γ–PKAholo complexes assembled with a truncated RIIα Δ44–86 construct using ISAC (Yang et al., 2012). (G) Projection averages of AKAP18γ–PKAholo complex assembled an RIIα ZeChimera construct. Scale bars in (F) and (G) represent 25 nm. (H) Statistical analysis of particle radius in angstroms (Å) for each AKAP18γ–PKAholo complexes. Box plot displays second and third quartile values, tails corresponding to minimum and maximum distances, (**) indicates p<0.01; (****) indicates p<0.0001.DOI:http://dx.doi.org/10.7554/eLife.01319.008
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fig3: Flexibility within RIIα constrains the configuration of the anchored kinase assembly.(A) Amino acid sequence alignment of the linker region in RIIα that connects the conserved N-terminal D/D domain to the C-terminal autoinhibitor and cAMP binding domains. This region shows low sequence homology and is likely to be structurally disordered. (B) Schematic representations of modified AKAP18γ–PKAholo complexes with AKAP18γ depicted in yellow, RIIα in green and PKAc in blue. (C) Biochemical analysis of the purified AKAP18γ–RIIα-PKAc complexes assembled with the wild-type RIIα subunit, the RIIα Δ44–86 mutant where the linker region was deleted, and the RIIα ZeChimera mutant where the mouse RIIα linker region was replaced with the corresponding and extended sequence from zebrafish. Top panel shows SDS-PAGE and Coomassie blue staining of the protein components. The next three panels show western blotting for RIIα, PKAc subunit and AKAP18γ, respectively. (D) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα Δ44–86 construct. (E) Electron micrograph of negatively stained AKAP18γ–PKAholo complexes (circles) assembled with the RIIα ZeChimera construct. Insets in (D and E) show enlarged views of individual particles outlined in gold for clarity. (F) Projection averages of the AKAP18γ–PKAholo complexes assembled with a truncated RIIα Δ44–86 construct using ISAC (Yang et al., 2012). (G) Projection averages of AKAP18γ–PKAholo complex assembled an RIIα ZeChimera construct. Scale bars in (F) and (G) represent 25 nm. (H) Statistical analysis of particle radius in angstroms (Å) for each AKAP18γ–PKAholo complexes. Box plot displays second and third quartile values, tails corresponding to minimum and maximum distances, (**) indicates p<0.01; (****) indicates p<0.0001.DOI:http://dx.doi.org/10.7554/eLife.01319.008
Mentions: Our model of the anchored PKA complex implies that an intrinsically disordered flexible linker region within RIIα supports the array of conformations that were observed in the raw micrographs and the projection averages (Figures 1 and 2C). To quantitatively assess the distribution of conformations assumed by this complex, we measured the end-to-end distance between the two large peripheral densities of 223 individual particles (Figure 2D). This population of structures followed a Gaussian distribution (Figure 2D, green trace) with a mean particle length of 275 ± 65 Å (n = 223). We propose that conformational plasticity observed in these analyses is facilitated by this intrinsically disordered region between residues 44 and 90 of RIIα, a linker that connects the AKAP docking site (D/D domain) to the cAMP-responsive transduction domains. This notion is further substantiated by a primary sequence analysis of RIIα orthologs, showing that the linker regions are of similar length but exhibit low amino acid identity (Figure 3A).10.7554/eLife.01319.008Figure 3.Flexibility within RIIα constrains the configuration of the anchored kinase assembly.

Bottom Line: Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits.Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates.We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, United States.

ABSTRACT
Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.

Show MeSH
Related in: MedlinePlus