2017 Jun 23
Local Protein Kinase A Action Proceeds Through Intact Holoenzymes
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Local Protein Kinase A Action Proceeds Through Intact Holoenzymes
Hormones can transmit signals through adenosine 3',5'-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) subunits with A-kinase-anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology- and live cell-imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated.
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Fig. 1. AKAP-PKA holoenzyme assemblies remain intact upon cAMP stimulation
A) SDS—polyacrylamide gel electrophoresis (SDS-PAGE) and stain-free visualization of a purified complex of AKAP79 with RII and C subunits of PKA. ( B) Stain-free visualization on native gels of intact AKAP79-PKA holoenzyme complex. ( C to E) Representative negative-stain EM micrographs of AKAP79:2RII:2C single particles. Particles were low-pass filtered to 20 Å. (D) Traces outlining the selected particles in (C). (E) Reference-free class averages from RELION 2D alignment and classification showing range of particle lengths. ( F and G) Schematics illustrating the flexibility and range of motion in AKAP79-anchored PKA holoenzymes. A compact state may favor the phosphorylation of associated substrates (F), whereas an extended conformation may allow PKA to act on multiple distinct local substrates (G). ( H) Zero-charge state native nanoelectrospray ionization mass spectrum of RII+C+AKAP (297–427) complexes. The stoichiometry of AKAP79(297–427) (grey), RII (green), and C subunit (orange) is indicated. ( I) Percentage of C subunit bound to AKAP79(297–427)—PKA complexes after incubation with increasing cAMP concentrations. Data are presented as means ± SEM. (Inset) Coomassie blue staining of proteins in pull-down experiments.
Fig. 2. Anchored PKA holoenzyme dynamics upon ligand stimulation
Western blot analysis of PKA C and RII subunits in (
A) AKAP79 or ( B) AKAP18g immunoprecipitates (IPs) after isoproterenol stimulation (Iso). ( C) IP and Western blot analysis of AKAP79 complexes isolated in the presence of phosphodiesterase inhibitors. ( D) Schematic depicting RII-CFP and C-YFP intermolecular FRET. ( E to H) FRET analysis of holoenzyme dissociation in HEK293 cells. (E) Time course of representative cells (0 to 400 s) stimulated with isoproterenol. (F) Iso + PDE4 inhibitor rolipram. (G) Iso + PDE3 inhibitor milrinone. (H) Amalgamated data from 25 recordings under each experimental condition. ( I) Schematic depicting ICUE3 cAMP FRET sensor. ( J) ICUE3 FRET recordings show cAMP production in response to Iso (black), Iso + rolipram (red), and forskolin (blue). The number of experiments is indicated. ( K and L) FRET analysis upon two trains of hormonal stimulation. (K) Intermolecular FRET analysis of holoenzyme dissociation in HEK293 cells upon two trains of stimulation with isoproterenol (black) and Iso + PDE4 inhibitor rolipram (red). (L) Monitoring cAMP accumulation with ICUE3 FRET sensor in response to isoproterenol (black) and Iso + PDE4 inhibitor rolipram (red). Amalgamated data from 25 recordings under each experimental condition. ( M to O) Proximity ligation assay (PLA) signal-intensity projections show RII-C interactions in (M) Unstimulated, (N) Iso-stimulated, and (O) Iso- and rolipram-treated HEK293 cells. ( P) Quantification of PLA puncta per cell using Fiji/ImageJ. All data are presented as means ± SEM.
Fig. 3. CRISPR-Cas9 PKA triple-KO and rescue with an RII-C fusion
A) Schematic depicting the R2C2 PKA fusion enzyme. ( B) Immunoblots confirming knockout of PKA subunit proteins: (Top) RIIα, (top middle) RIIβ, ανδ (bottom middle) C subunit. (Bottom) Ponceau staining shows equal loading in WT and U2OS cells. ( R2A/R2B/CA C) Western blots for PKA C subunit confirm expression of R2C2 fusion. ( D) Schematic of rapamycin induced heterodimerization of PKA subunits. ( E) Immunoblots showing that rapamycin chemically locks the PKA holoenzyme at supraphysiological concentrations of cAMP. ( F) Schematic of AKAR4 PKA activity biosensor. ( G) Cytoplasmic FRET recordings in response to Iso (1 μM) stimulation in U2OS cells expressing RII-FKBP and C-FRB in the presence (blue) or absence (orange) of rapamycin (100 nM). ( R2A/R2B/CA H) Representative cells showing cytoplasmic AKAR4 FRET response upon rescue with (left) RIIα and Cα and (right) R2C2 fusion. ( I) Cytoplasmic FRETrecordings in triple-KO U2OS cells (gray) and cells rescued with R2C2 fusion (green) or WT RIIα and Cα (orange). ( R2A/R2B/CA J) Montage of cells showing nuclear AKAR4 FRET signals upon rescue with (left) RIIα and Cα or (right) R2C2 fusion. ( K) Nuclear FRET recordings in U2OS cells (gray) and cells rescued with R2C2 fusion (green) or WT RIIα and Cα (orange). ( R2A/R2B/CA L) Proliferation of triple KO U2OS cells rescued with R2C2 fusion (blue) or two different clones of quadruple KO U2OS R2A/R2B/CA cells rescued with R2C2 fusion (red and green). R2A/R2B/CA/CB
Fig. 4. Chemical-genetic and pharmacological control of mitochondrial PKA action
A) Schematic depicting the introduction of an analog-sensitive kinase mutation into the R2C2 PKA fusion enzyme. The activity of this pharmacologically controlled fusion kinase was monitored in U2OS cells. ( R2A/R2B/CA B) Representative cells showing cytoplasmic AKAR FRET signals at selected time points from cells expressing (top) R2C2 and (bottom) the 1-NM-PP1—sensitive R2C2 M120A mutant. The left image in both panels shows expression of the FRET reporter. The others panels show pseudocolor images of the FRET intensity at one time point before stimulation (−45 s), one time point after stimulation with isoproterenol (105 s), and a third time point after addition of 1-NM-PP1 (415 s). ( C) Time courses of cytoplasmic AKAR4 FRET responses to Iso (1 μM, time zero) and 1-NM-PP1 (2 μM, 300 s) in PKA triple-KO cells expressing the (green) R2C2 fusion or (blue) the R2C2 M120A mutant. ( D) Schematic depicting protective effects of anchored PKA due to phosphorylation of BAD and the resultant release of the prosurvival BCL-2 protein. ( E) Inhibition of PKA directed BAD pSer 155 phosphorylation by 1-NM-PP1 in cells expressing R2C2 M120A. Immunoblot detecting (top) BAD pSer 155, (middle) R2C2, and (bottom) GAPDH loading controls. ( F) Quantification of BAD pSer 155 signal from immunoblots from three independent experiments. Data are presented as means ± SEM. ( G) Fluorescent detection of apoptotic events upon treatment of cells with the apoptotic agent etoposide, plus and minus β-agonists and 1-NM-PP1. Apoptotic cells were detected by (green) activation of caspase and (magenta) condensation of DNA. ( H) Quantification of the percentage of apoptotic cells from ( G). The numbers of cells monitored are indicated above each column. Data are presented as means ± SEM.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
A Kinase Anchor Proteins / metabolism
Cyclic AMP-Dependent Protein Kinases / chemistry
Cyclic AMP-Dependent Protein Kinases / genetics
Cyclic AMP-Dependent Protein Kinases / metabolism
Holoenzymes / metabolism
Mitochondria / enzymology
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Recombinant Fusion Proteins
Cyclic AMP-Dependent Protein Kinases
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