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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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Comparison of AKAR-18RBS and AKAR-18RBS–pro anchoring and FRET controls.(A) Representative confocal micrographs of live cells show comparable expression levels and the intracellular localization of AKAR-18RBS (green) and mCherry-RIIα (red). Composite images are also shown. HEK293 cells were transiently transfected with AKAR-18RBS and either PKA RIIα wild type (upper panels), RIIα Δ 44–86 (middle panels) or RIIα ZeChimera (lower panels). Scale bars represent 5 μm. (B) Schematic of AKAR-18RBS–pro that contains two helix-breaking proline substitutions in the PKA anchoring site that abolish RII binding. (C) AKAR-18RBS co-precipitates all three RIIα variants as well as endogenous PKA catalytic subunit from transiently transfected HEK293 cells (top panels). AKAR-18RBS–pro no longer anchors RII and does not co-precipitate RIIα or PKAc (top panels). Control immunoblots show equivalent levels of AKAR-18RBS and AKAR-18RBS–pro in immunoprecipitates as well as RIIα and PKAc expression in cell lysates. (D) FRET recordings using AKAR-18RBS–pro. Traces show Iso stimulated changes in FRET in each cohort (0–400 s). Data are normalized to unstimulated basal FRET level for each respective RIIα form. The increase in the AKAR-18RBS normalized FRET ratio in cells co-expressing RIIα Δ44–86 (Figure 5H) is no longer present when the reporter is unable to bind PKA.DOI:http://dx.doi.org/10.7554/eLife.01319.012
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fig5s1: Comparison of AKAR-18RBS and AKAR-18RBS–pro anchoring and FRET controls.(A) Representative confocal micrographs of live cells show comparable expression levels and the intracellular localization of AKAR-18RBS (green) and mCherry-RIIα (red). Composite images are also shown. HEK293 cells were transiently transfected with AKAR-18RBS and either PKA RIIα wild type (upper panels), RIIα Δ 44–86 (middle panels) or RIIα ZeChimera (lower panels). Scale bars represent 5 μm. (B) Schematic of AKAR-18RBS–pro that contains two helix-breaking proline substitutions in the PKA anchoring site that abolish RII binding. (C) AKAR-18RBS co-precipitates all three RIIα variants as well as endogenous PKA catalytic subunit from transiently transfected HEK293 cells (top panels). AKAR-18RBS–pro no longer anchors RII and does not co-precipitate RIIα or PKAc (top panels). Control immunoblots show equivalent levels of AKAR-18RBS and AKAR-18RBS–pro in immunoprecipitates as well as RIIα and PKAc expression in cell lysates. (D) FRET recordings using AKAR-18RBS–pro. Traces show Iso stimulated changes in FRET in each cohort (0–400 s). Data are normalized to unstimulated basal FRET level for each respective RIIα form. The increase in the AKAR-18RBS normalized FRET ratio in cells co-expressing RIIα Δ44–86 (Figure 5H) is no longer present when the reporter is unable to bind PKA.DOI:http://dx.doi.org/10.7554/eLife.01319.012

Mentions: To follow these in vitro studies, we tested our hypothesis that flexibility within the anchored PKA holoenzyme influences cAMP signaling in living cells. We generated a modified fluorescence resonance energy transfer (FRET) based PKA activity sensor using the A-kinase activity reporter (AKAR2) backbone (Zhang et al., 2005). Our modified sensor (AKAR-18RBS) was constructed by fusing the PKA binding helix of AKAP18 (18RBS) (Fraser et al., 1998; Gray et al., 1998) to the amino terminus of AKAR2 (Figure 5A). This genetically encoded reporter detects PKA phosphorylation in real-time by monitoring changes in the YFP/CFP emission ratio inside cells (Figure 5A). As a prelude to these studies AKAR-18RBS association with wild-type RIIα or either of the modified RIIα constructs was confirmed by co-immunoprecipitation of each complex from HEK293 cells (Figure 5B). In parallel, immunoblot and confocal fluorescent imaging analyses confirmed that mCherry tagged versions of each RIIα form were expressed to equivalent levels (Figure 5C) and uniformly distributed in HEK293 cells (Figure 5—figure supplement 1A).10.7554/eLife.01319.011Figure 5.Flexibility within the anchored PKA holoenzyme impacts cAMP responsive signaling inside cells.


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)

Comparison of AKAR-18RBS and AKAR-18RBS–pro anchoring and FRET controls.(A) Representative confocal micrographs of live cells show comparable expression levels and the intracellular localization of AKAR-18RBS (green) and mCherry-RIIα (red). Composite images are also shown. HEK293 cells were transiently transfected with AKAR-18RBS and either PKA RIIα wild type (upper panels), RIIα Δ 44–86 (middle panels) or RIIα ZeChimera (lower panels). Scale bars represent 5 μm. (B) Schematic of AKAR-18RBS–pro that contains two helix-breaking proline substitutions in the PKA anchoring site that abolish RII binding. (C) AKAR-18RBS co-precipitates all three RIIα variants as well as endogenous PKA catalytic subunit from transiently transfected HEK293 cells (top panels). AKAR-18RBS–pro no longer anchors RII and does not co-precipitate RIIα or PKAc (top panels). Control immunoblots show equivalent levels of AKAR-18RBS and AKAR-18RBS–pro in immunoprecipitates as well as RIIα and PKAc expression in cell lysates. (D) FRET recordings using AKAR-18RBS–pro. Traces show Iso stimulated changes in FRET in each cohort (0–400 s). Data are normalized to unstimulated basal FRET level for each respective RIIα form. The increase in the AKAR-18RBS normalized FRET ratio in cells co-expressing RIIα Δ44–86 (Figure 5H) is no longer present when the reporter is unable to bind PKA.DOI:http://dx.doi.org/10.7554/eLife.01319.012
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fig5s1: Comparison of AKAR-18RBS and AKAR-18RBS–pro anchoring and FRET controls.(A) Representative confocal micrographs of live cells show comparable expression levels and the intracellular localization of AKAR-18RBS (green) and mCherry-RIIα (red). Composite images are also shown. HEK293 cells were transiently transfected with AKAR-18RBS and either PKA RIIα wild type (upper panels), RIIα Δ 44–86 (middle panels) or RIIα ZeChimera (lower panels). Scale bars represent 5 μm. (B) Schematic of AKAR-18RBS–pro that contains two helix-breaking proline substitutions in the PKA anchoring site that abolish RII binding. (C) AKAR-18RBS co-precipitates all three RIIα variants as well as endogenous PKA catalytic subunit from transiently transfected HEK293 cells (top panels). AKAR-18RBS–pro no longer anchors RII and does not co-precipitate RIIα or PKAc (top panels). Control immunoblots show equivalent levels of AKAR-18RBS and AKAR-18RBS–pro in immunoprecipitates as well as RIIα and PKAc expression in cell lysates. (D) FRET recordings using AKAR-18RBS–pro. Traces show Iso stimulated changes in FRET in each cohort (0–400 s). Data are normalized to unstimulated basal FRET level for each respective RIIα form. The increase in the AKAR-18RBS normalized FRET ratio in cells co-expressing RIIα Δ44–86 (Figure 5H) is no longer present when the reporter is unable to bind PKA.DOI:http://dx.doi.org/10.7554/eLife.01319.012
Mentions: To follow these in vitro studies, we tested our hypothesis that flexibility within the anchored PKA holoenzyme influences cAMP signaling in living cells. We generated a modified fluorescence resonance energy transfer (FRET) based PKA activity sensor using the A-kinase activity reporter (AKAR2) backbone (Zhang et al., 2005). Our modified sensor (AKAR-18RBS) was constructed by fusing the PKA binding helix of AKAP18 (18RBS) (Fraser et al., 1998; Gray et al., 1998) to the amino terminus of AKAR2 (Figure 5A). This genetically encoded reporter detects PKA phosphorylation in real-time by monitoring changes in the YFP/CFP emission ratio inside cells (Figure 5A). As a prelude to these studies AKAR-18RBS association with wild-type RIIα or either of the modified RIIα constructs was confirmed by co-immunoprecipitation of each complex from HEK293 cells (Figure 5B). In parallel, immunoblot and confocal fluorescent imaging analyses confirmed that mCherry tagged versions of each RIIα form were expressed to equivalent levels (Figure 5C) and uniformly distributed in HEK293 cells (Figure 5—figure supplement 1A).10.7554/eLife.01319.011Figure 5.Flexibility within the anchored PKA holoenzyme impacts cAMP responsive signaling inside cells.

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