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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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Purification and electron microscopy of the AKAP18γ–PKAholo complex.(A) SDS-PAGE and Coomassie staining of purified individual complex components. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractions at the leading edge of the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel electrophoresis (right) obtained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo complexes (circles). Triangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arrow) conjugated to an AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asterisks in D). (E) Right, highlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct conformations using ISAC (Yang et al., 2012). Unlabeled scale bars represent 25 nm.DOI:http://dx.doi.org/10.7554/eLife.01319.003
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fig1: Purification and electron microscopy of the AKAP18γ–PKAholo complex.(A) SDS-PAGE and Coomassie staining of purified individual complex components. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractions at the leading edge of the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel electrophoresis (right) obtained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo complexes (circles). Triangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arrow) conjugated to an AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asterisks in D). (E) Right, highlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct conformations using ISAC (Yang et al., 2012). Unlabeled scale bars represent 25 nm.DOI:http://dx.doi.org/10.7554/eLife.01319.003

Mentions: Macromolecular assemblies were formed when purified human γ isoform of AKAP18 (AKAP18γ) was incubated with the PKA holoenzyme (PKAholo), formed by the RIIα and PKAc subunits (Figure 1A, ‘Materials and methods’). Following separation by size-exclusion chromatography, fractions from the front of the elution peak (indicated by arrow in Figure 1B) were analyzed by SDS-PAGE (Figure 1C, left). The subunit composition was confirmed by immunoblotting (Figure 1C, mid). Native electrophoresis established that a majority of this material migrated as a single species with an apparent molecular weight in excess of 240 kD (Figure 1C, right). This molecular mass is consistent with a hetero-pentameric complex composed of a single AKAP18γ molecule anchored to an RIIα subunit dimer and two PKAc subunits.10.7554/eLife.01319.003Figure 1.Purification and electron microscopy of the AKAP18γ–PKAholo complex.


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)

Purification and electron microscopy of the AKAP18γ–PKAholo complex.(A) SDS-PAGE and Coomassie staining of purified individual complex components. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractions at the leading edge of the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel electrophoresis (right) obtained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo complexes (circles). Triangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arrow) conjugated to an AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asterisks in D). (E) Right, highlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct conformations using ISAC (Yang et al., 2012). Unlabeled scale bars represent 25 nm.DOI:http://dx.doi.org/10.7554/eLife.01319.003
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Related In: Results  -  Collection

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fig1: Purification and electron microscopy of the AKAP18γ–PKAholo complex.(A) SDS-PAGE and Coomassie staining of purified individual complex components. (B) Size-exclusion chromatography (SEC) trace for purification of the assembled AKAP18γ–PKAholo complex. Fractions at the leading edge of the peak (indicated by gray bar) were chosen for further analysis. (C) SDS-PAGE (left), western blot (middle) and native gel electrophoresis (right) obtained from the SEC peak elution fraction (arrow in B). (D) Electron micrograph of the negatively stained AKAP18γ–PKAholo complexes (circles). Triangles indicate the three major densities of the AKAP18γ–PKAholo complex. Inset, shows labeling with a gold nanoparticle (arrow) conjugated to an AKAP18γ-streptavidin moiety (arrow). (E) Left, enlarged images of individual AKAP18γ–PKAholo complexes (denoted by asterisks in D). (E) Right, highlighted outline (yellow) of particle shapes. (F) Projection averages of the AKAP18γ–PKAholo complex classified into distinct conformations using ISAC (Yang et al., 2012). Unlabeled scale bars represent 25 nm.DOI:http://dx.doi.org/10.7554/eLife.01319.003
Mentions: Macromolecular assemblies were formed when purified human γ isoform of AKAP18 (AKAP18γ) was incubated with the PKA holoenzyme (PKAholo), formed by the RIIα and PKAc subunits (Figure 1A, ‘Materials and methods’). Following separation by size-exclusion chromatography, fractions from the front of the elution peak (indicated by arrow in Figure 1B) were analyzed by SDS-PAGE (Figure 1C, left). The subunit composition was confirmed by immunoblotting (Figure 1C, mid). Native electrophoresis established that a majority of this material migrated as a single species with an apparent molecular weight in excess of 240 kD (Figure 1C, right). This molecular mass is consistent with a hetero-pentameric complex composed of a single AKAP18γ molecule anchored to an RIIα subunit dimer and two PKAc subunits.10.7554/eLife.01319.003Figure 1.Purification and electron microscopy of the AKAP18γ–PKAholo complex.

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