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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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Phosphorylation of PDE4D is time-dependent.AKAP18 complexes formed with either RIIα or each variant were incubated with PDE4D3 and γ-32P-ATP for the times indicated. (Top panel) Autoradiograph showing incorporation of 32P into PDE4D in each complex. (Middle and bottom panels) India ink staining of the membrane, showing components of the assay. Total PDE4D3 is in the middle, and RII, PKAc and AKAP18γ are shown in the bottom panel.DOI:http://dx.doi.org/10.7554/eLife.01319.010
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fig4s1: Phosphorylation of PDE4D is time-dependent.AKAP18 complexes formed with either RIIα or each variant were incubated with PDE4D3 and γ-32P-ATP for the times indicated. (Top panel) Autoradiograph showing incorporation of 32P into PDE4D in each complex. (Middle and bottom panels) India ink staining of the membrane, showing components of the assay. Total PDE4D3 is in the middle, and RII, PKAc and AKAP18γ are shown in the bottom panel.DOI:http://dx.doi.org/10.7554/eLife.01319.010

Mentions: In the second phase, experiments were conducted using higher-order complexes formed with wild-type RIIα, RIIα Δ44–86, or the RIIα ZeChimera (assembled as described above) to investigate whether manipulating the intrinsic flexibility of PKA altered phosphorylation of anchored PDE4D (Figure 4F–G). We measured cAMP-independent phosphorylation of PDE4D at 5 min, a time point that showed sub-maximal substrate phosphorylation (Figure 4F,G, Figure 4—figure supplement 1). Basal PDE4D phosphorylation was enhanced 1.97 ± 0.18-fold (n = 6, p<0.05) in complexes formed with RIIα Δ44–86 when compared to a wild-type complex (Figure 4G, bars 1 and 4). In contrast, extension of the linker region in the context of AKAP18γ–RIIα ZeChimera PKAholo assembly had no effect as compared to wild type (Figure 4G, bars 1 and 7). Control experiments confirmed that addition of cAMP further augmented phosphorylation of PDE4D in all cases (Figure 4F, lanes 2, 5 and 8) and pretreatment with PKI inhibitor peptide abolished anchored kinase activity (Figure 4F, lanes 3, 6 and 9). These data show that the AKAP can be thought of as a catalyst that physically brings the reactants together, and the flexibility within the anchored PKA holoenzyme allows for the precise orientation of the enzyme and substrate. This mechanism may be particularly relevant for cAMP-independent phosphorylation events that are believed to represent approximately 30% of PKA action (Taylor et al., 2012). Therefore, this hitherto unexplained but critical component of cellular PKA activity may be accomplished by the persistent phosphorylation of substrates embedded in higher-order AKAP signaling assemblies.


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)

Phosphorylation of PDE4D is time-dependent.AKAP18 complexes formed with either RIIα or each variant were incubated with PDE4D3 and γ-32P-ATP for the times indicated. (Top panel) Autoradiograph showing incorporation of 32P into PDE4D in each complex. (Middle and bottom panels) India ink staining of the membrane, showing components of the assay. Total PDE4D3 is in the middle, and RII, PKAc and AKAP18γ are shown in the bottom panel.DOI:http://dx.doi.org/10.7554/eLife.01319.010
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814001&req=5

fig4s1: Phosphorylation of PDE4D is time-dependent.AKAP18 complexes formed with either RIIα or each variant were incubated with PDE4D3 and γ-32P-ATP for the times indicated. (Top panel) Autoradiograph showing incorporation of 32P into PDE4D in each complex. (Middle and bottom panels) India ink staining of the membrane, showing components of the assay. Total PDE4D3 is in the middle, and RII, PKAc and AKAP18γ are shown in the bottom panel.DOI:http://dx.doi.org/10.7554/eLife.01319.010
Mentions: In the second phase, experiments were conducted using higher-order complexes formed with wild-type RIIα, RIIα Δ44–86, or the RIIα ZeChimera (assembled as described above) to investigate whether manipulating the intrinsic flexibility of PKA altered phosphorylation of anchored PDE4D (Figure 4F–G). We measured cAMP-independent phosphorylation of PDE4D at 5 min, a time point that showed sub-maximal substrate phosphorylation (Figure 4F,G, Figure 4—figure supplement 1). Basal PDE4D phosphorylation was enhanced 1.97 ± 0.18-fold (n = 6, p<0.05) in complexes formed with RIIα Δ44–86 when compared to a wild-type complex (Figure 4G, bars 1 and 4). In contrast, extension of the linker region in the context of AKAP18γ–RIIα ZeChimera PKAholo assembly had no effect as compared to wild type (Figure 4G, bars 1 and 7). Control experiments confirmed that addition of cAMP further augmented phosphorylation of PDE4D in all cases (Figure 4F, lanes 2, 5 and 8) and pretreatment with PKI inhibitor peptide abolished anchored kinase activity (Figure 4F, lanes 3, 6 and 9). These data show that the AKAP can be thought of as a catalyst that physically brings the reactants together, and the flexibility within the anchored PKA holoenzyme allows for the precise orientation of the enzyme and substrate. This mechanism may be particularly relevant for cAMP-independent phosphorylation events that are believed to represent approximately 30% of PKA action (Taylor et al., 2012). Therefore, this hitherto unexplained but critical component of cellular PKA activity may be accomplished by the persistent phosphorylation of substrates embedded in higher-order AKAP signaling assemblies.

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