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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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Three-dimensional EM maps of the AKAP18γ–PKAholo complex.Rotated views of the 3D reconstructions for the AKAP18γ–PKAholo complex determined to 35 Å resolution. (A) In the triangular conformation, the three spherical densities are arranged in a pseudo-symmetric fashion, with the two peripheral densities extending 300 Å apart and at an angle of 100° with respect to the central density. The smaller central density is 60 × 60 × 80 Å and the two larger peripheral densities are 100 × 100 × 85 Å. (B) In the linear conformation, the three spherical densities are arranged in a linear fashion, with the two peripheral domains extending 385 Å apart. (C and D) Back-projections (right) of the calculated 3D maps are compared to class averages (left) used for generating the initial reconstructions in IMAGIC (van Heel et al., 1996).DOI:http://dx.doi.org/10.7554/eLife.01319.007
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fig2s2: Three-dimensional EM maps of the AKAP18γ–PKAholo complex.Rotated views of the 3D reconstructions for the AKAP18γ–PKAholo complex determined to 35 Å resolution. (A) In the triangular conformation, the three spherical densities are arranged in a pseudo-symmetric fashion, with the two peripheral densities extending 300 Å apart and at an angle of 100° with respect to the central density. The smaller central density is 60 × 60 × 80 Å and the two larger peripheral densities are 100 × 100 × 85 Å. (B) In the linear conformation, the three spherical densities are arranged in a linear fashion, with the two peripheral domains extending 385 Å apart. (C and D) Back-projections (right) of the calculated 3D maps are compared to class averages (left) used for generating the initial reconstructions in IMAGIC (van Heel et al., 1996).DOI:http://dx.doi.org/10.7554/eLife.01319.007

Mentions: Three-dimensional (3D) reconstructions for the triangular and linear configurations of the AKAP18γ–PKAholo complex were determined at 35 Å resolution from a tilted-series dataset (Figure 2A, Figure 2—figure supplements 1 and 2). Negative staining in EM can cause flattening of particles, which might lead to apparent structural distortions. However, inspection of particles at various tilt angles showed that particles of linear and triangular conformations remained clearly distinguishable, even at high tilt angles (Figure 2—figure supplement 1). In both 3D reconstructions, a central mass with dimensions of 60 × 60 × 80 Å (corresponding to the site of AKAP18γ as demonstrated by affinity gold labeling, Figure 2A, black triangle and Figure 1D, inset) was flanked on either side by larger densities of 100 × 100 × 85 Å (Figure 2A). These flanking densities can each accommodate a sub-complex of RIIα and PKAc. In the triangular conformation, the two peripheral densities are oriented at a 100° angle with respect to the central density and exhibit an end-to-end length of ∼300 Å (Figure 2A, top). The end-to-end length increases to ∼385 Å in the extended linear configuration (Figure 2A, bottom). Back-projections calculated from the final 3D maps compare well with the experimental class averages (Figure 2—figure supplement 2). Moreover, this back-projection analysis demonstrated that when the triangular model is tilted completely on its edge (where it may appear more linear in projection) its dimensions (max length = 300 Å) are significantly smaller than the maximum end-to-end length obtained for the linear reconstruction (max length = 385 Å). Hence, we conclude that the linear and triangular conformations are structurally distinct.10.7554/eLife.01319.005Figure 2.3D reconstructions and pseudo-atomic structure 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)

Three-dimensional EM maps of the AKAP18γ–PKAholo complex.Rotated views of the 3D reconstructions for the AKAP18γ–PKAholo complex determined to 35 Å resolution. (A) In the triangular conformation, the three spherical densities are arranged in a pseudo-symmetric fashion, with the two peripheral densities extending 300 Å apart and at an angle of 100° with respect to the central density. The smaller central density is 60 × 60 × 80 Å and the two larger peripheral densities are 100 × 100 × 85 Å. (B) In the linear conformation, the three spherical densities are arranged in a linear fashion, with the two peripheral domains extending 385 Å apart. (C and D) Back-projections (right) of the calculated 3D maps are compared to class averages (left) used for generating the initial reconstructions in IMAGIC (van Heel et al., 1996).DOI:http://dx.doi.org/10.7554/eLife.01319.007
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2s2: Three-dimensional EM maps of the AKAP18γ–PKAholo complex.Rotated views of the 3D reconstructions for the AKAP18γ–PKAholo complex determined to 35 Å resolution. (A) In the triangular conformation, the three spherical densities are arranged in a pseudo-symmetric fashion, with the two peripheral densities extending 300 Å apart and at an angle of 100° with respect to the central density. The smaller central density is 60 × 60 × 80 Å and the two larger peripheral densities are 100 × 100 × 85 Å. (B) In the linear conformation, the three spherical densities are arranged in a linear fashion, with the two peripheral domains extending 385 Å apart. (C and D) Back-projections (right) of the calculated 3D maps are compared to class averages (left) used for generating the initial reconstructions in IMAGIC (van Heel et al., 1996).DOI:http://dx.doi.org/10.7554/eLife.01319.007
Mentions: Three-dimensional (3D) reconstructions for the triangular and linear configurations of the AKAP18γ–PKAholo complex were determined at 35 Å resolution from a tilted-series dataset (Figure 2A, Figure 2—figure supplements 1 and 2). Negative staining in EM can cause flattening of particles, which might lead to apparent structural distortions. However, inspection of particles at various tilt angles showed that particles of linear and triangular conformations remained clearly distinguishable, even at high tilt angles (Figure 2—figure supplement 1). In both 3D reconstructions, a central mass with dimensions of 60 × 60 × 80 Å (corresponding to the site of AKAP18γ as demonstrated by affinity gold labeling, Figure 2A, black triangle and Figure 1D, inset) was flanked on either side by larger densities of 100 × 100 × 85 Å (Figure 2A). These flanking densities can each accommodate a sub-complex of RIIα and PKAc. In the triangular conformation, the two peripheral densities are oriented at a 100° angle with respect to the central density and exhibit an end-to-end length of ∼300 Å (Figure 2A, top). The end-to-end length increases to ∼385 Å in the extended linear configuration (Figure 2A, bottom). Back-projections calculated from the final 3D maps compare well with the experimental class averages (Figure 2—figure supplement 2). Moreover, this back-projection analysis demonstrated that when the triangular model is tilted completely on its edge (where it may appear more linear in projection) its dimensions (max length = 300 Å) are significantly smaller than the maximum end-to-end length obtained for the linear reconstruction (max length = 385 Å). Hence, we conclude that the linear and triangular conformations are structurally distinct.10.7554/eLife.01319.005Figure 2.3D reconstructions and pseudo-atomic structure 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