SIGNAL TRANSDUCTION

Local protein kinase A actionproceeds through intact holoenzymesF. Donelson Smith,1 Jessica L. Esseltine,1* Patrick J. Nygren,1 David Veesler,2

Dominic P. Byrne,3 Matthias Vonderach,3 Ilya Strashnov,4 Claire E. Eyers,3

Patrick A. Eyers,3 Lorene K. Langeberg,1 John D. Scott1†

Hormones can transmit signals through adenosine 3ʹ,5ʹ-monophosphate (cAMP) toprecise intracellular locations. The fidelity of these responses relies on the activation oflocalized 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 thatAKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets.Native mass spectrometry established that these macromolecular assembliesincorporated stoichiometric amounts of cAMP. Chemical-biology– and live cell–imagingtechniques revealed that catalytically active PKA holoenzymes remained intact within thecytoplasm. These findings indicate that the parameters of anchored PKA holoenzymeaction are much more restricted than originally anticipated.

Classical in vitro biochemistry and elegantstructural studies have led to a commonlyheld view that the active protein kinase A(PKA) catalytic (C) subunit dissociates fromthe regulatory subunit dimer in the pres-

ence of excess adenosine 3′,5′-monophosphate(cAMP) (1–3). Yet the utility of this rudimentarymechanism inside cells remains unclear (4). Single-particle negative-stain electron microscopy (EM)analysis of A-kinase–anchoring protein 79 withPKA (AKAP79:PKA) holoenzyme complexes ofAKAP79, the regulatory II (RII) subunit, and theC subunit (AKAP79:2RII:2C) detected a range ofconformationally distinct species (Fig. 1, A to D,and fig. S1). A set of ~14,000 particles was sub-jected to reference-free two-dimensional (2D) clas-sification using RELION (5). Poorly aligned classeswere discarded over multiple iterations to revealan ensemble of three-lobed assemblies in whichthe peripheral densities are observed at differentdistances fromeach other. These range from 150Åin the compact configuration to 250 Å in the ex-tended conformation (Fig. 1E). The tandem pe-ripheral lobes represent the C subunit in complexwith the cAMP-binding domains of RII (6), whereasthe central density represents the AKAP-RII di-mer interface (Fig. 1E). Thus, AKAP79 constrainseach subassembly of regulatory and C subunitsto within 250 Å of substrates. We surmise thatthe restricted movement of the anchored C sub-unit in this configuration augments phosphoryl-ation of local substrates that are either interacting

with the AKAP (Fig. 1F) or in proximity to theperipheral lobes of the extended PKA holoenzyme(Fig. 1G).An implication of our structural model is that,

although local cAMP production stimulates ki-nase activity, AKAP79:2RII:2C assemblies canremain intact. Native mass spectrometry (MS)allows the measurement of molecular mass andis used to determine the stoichiometry of intactprotein complexes in the gas phase (7–9). In thepresence of cAMP, which copurifies with RII, weobserved higher-order complexes, including theintact 2RII:2C PKA holoenzyme and the pen-tameric AKAP79:2RII:2C assembly (Fig. 1H, fig. S1,and tables S1 and S2). From these experiments,we infer a minimal disruption of the AKAP-PKAarchitecture even in the presence of cAMP.To address the relative stability of the pentame-

ric AKAP79:2RII:2C assembly in the presence ofelevated cAMP, we performed pull-down experi-ments with a fragment of the anchoring proteinAKAP79297–427. Retention of anchored C subunitsin the presence of increasing concentrations ofcAMPwas assessed by quantification of Coomassieblue–stained protein (Fig. 1I and inset). At physi-ological concentrations of cAMP (1 to 2 mM) mostof the C subunit (~70 to 80%) remained associatedwith the AKAP79297–427-RII complex (Fig. 1I andinset). Substantial release of the C subunit wasonly evident at supraphysiological levels of cAMP(Fig. 1I; 10 to 90 mM), and no change in bindingof RII to AKAP79297–427 was observed. In controlexperiments, 5′-AMP (100 mM), a degradationproduct of cAMP, did not alter anchored holo-enzyme composition (Fig. 1I). Thus, physiologicalamounts of cAMP promote minimal release ofthe C subunit from the anchored holoenzymein vitro.Additional studies used high-resolution native

MS to evaluate cAMP occupancy in PKA holo-enzyme complexes. At basal cAMP concentrations,multiple charge states of 2RII:C subcomplexeswere

observed betweenmass/charge ratios of 5700 and6300. These species preferentially contained two,but up to four, molecules of cAMP (fig. S1). Whenanalysiswasdoneathigher concentrationsof cAMP(5 mM), the predominant species exhibited cyclicnucleotide occupancy of 4mol per RII dimer (fig.S1 and table S3). Notably, the C subunit remainedattached under these conditions (fig. S1). Thus,we can conclude that a substantial proportionof the PKA C subunit remained associated withtheAKAP79297–427-RII dimer when cAMP-bindingsites were occupied.For amore stringent in situ test, wemonitored

the integrity of cellular AKAP-PKA complexes inresponse to ligand activation. AKAP79 or AKAP18gcomplexes were immunoprecipitated from celllysates prepared after stimulation of cells withthe b-adrenergic agonist isoproterenol (Iso, 1 mM).Immunoblot analysis detected equivalent amountsof C subunit in samples from cells stimulated withisoproterenol or vehicle control (Fig. 2, A and B,top, lane 2). Local cAMP flux is controlled througha balance of second-messenger production byadenylyl cyclases and degradation by phospho-diesterases (PDEs) (10). Cells were stimulatedwithisoproterenol in the presence of the general PDEinhibitor 3-isobutyl-1-methylxanthine (IBMX), theselective PDE3 inhibitor milrinone, or the PDE4-specific inhibitor rolipram (11, 12). The compositionof AKAP79-PKA complexes was assessed by im-munoblot. Intact complexes were detected in con-trol and Iso-treated samples (Fig. 2C, lanes 1 and2). Inclusion of rolipram promoted full dissocia-tion of the C subunit (Fig. 2C, lane 3). Likewise,application of IBMX induced release of the C sub-unit from AKAP complexes (Fig. 2C, lane 5). Incontrast, inclusion of milrinone had little effect,indicating that PDE3 is nonfunctional in theseAKAP79-PKA microdomains (Fig. 2C, lane 4). Ki-nase activation was monitored by immunoblotdetection of phospho-PKA substrates (Fig. 2C,bottom panel). Similar findings were obtainedupon analysis of AKAP18g complexes and whenprostaglandin E1 (PGE1) or epinephrine was usedas an agonist (fig. S2). Treatment with rolipramalone did not activate PKA or release C subunits(Fig. 2C, lane 6). Thus, native production of cAMPin response to physiological effectors of GPCRsignaling appears not to promote C subunit releasefrom anchored PKA holoenzymes.Förster resonance energy transfer (FRET) was

used to investigate PKAholoenzyme compositionin real time (13). Initially, we usedRII conjugatedto cyan fluorescent protein (RII-CFP) and the Csubunit conjugated to yellow fluorescent pro-tein (C-YFP) as intermolecular FRET probes tomonitor the integrity of the PKA holoenzyme af-ter agonist stimulation of cells (Fig. 2D). Isopro-terenol promoted minimal change in the CFP/YFP FRET ratio over time courses of 400 s (Fig.2, E and H, black), consistent with negligibledissociation of the PKA holoenzyme. In contrast,preincubation of cells with rolipram before stim-ulation with b-adrenergic agonist triggered apronounced and time-dependent reduction inthe FRET ratio (Fig. 2, F and H, red). Thus, dis-sociation of the PKA holoenzyme only occurred

RESEARCH

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 1 of 6

1Howard Hughes Medical Institute, Department ofPharmacology, University of Washington, Seattle, WA 98195,USA. 2Department of Biochemistry, University of Washington,Seattle, WA 98195, USA. 3Department of Biochemistry,Institute of Integrative Biology, University of Liverpool,Liverpool L69 7ZB, UK. 4School of Chemistry, The Universityof Manchester, Manchester M13 9PL, UK.*Present address: Schulich School of Medicine and Dentistry,Western University, London, Ontario N6A 5C1, Canada.†Corresponding author. Email: scottjdw@uw.edu

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if cAMP accumulated to supraphysiological con-centrations. Pretreatment of cells with the PDE3inhibitor milrinone had little effect on the FRETresponse (Fig. 2, G andH, gray). Comparable FRETrecordings were obtained when PGE1 was usedas the agonist (fig. S2).The ICUE3 FRET biosensor detects agonist-

responsive accumulation of cAMP (Fig. 2I) (14).Stimulation of cells with isoproterenol or PGE1promoted transient increases in the FRET signal(Fig. 2J, black, and fig. S2). This response wasenhanced by addition of rolipram (Fig. 2J, red,and fig. S2). However, the highest amounts ofcAMP production (15 times physiological levels)were recorded upon application of forskolin, adirect activator of adenylyl cyclases and a com-mon tool in cAMP research (Fig. 2J, blue). Thus,forskolin sustains supraphysiological accumula-tion of cAMP to concentrations far above thoseinduced by b-agonists.We investigated the effect of recurrent stimu-

lation of cAMP synthesis on PKA holoenzymeintegrity with the same biosensors. After an ini-tial pulse of isoproterenol, the drug was washedout for 500 s before application of a secondstimulus (Fig. 2, K and L). Intermolecular FRETrevealed that RII-CFP and C-YFP remained inproximity over the duration of these experiments(Fig. 2K, black). As expected, costimulation ofcells with isoproterenol and rolipram initiatedcycles of holoenzyme dissociation and reforma-tion (Fig. 2K, red). Complementary biochemicalexperiments revealed that washout of rolipramand isoproterenol promoted reformation ofAKAP79-PKA holoenzyme over a similar timecourse (fig. S2). Using the ICUE3 biosensor, wedemonstrated that isoproterenol induces a tran-sient rise in the cellular concentration of cAMP thatapproachesbaselineduringwashout (Fig. 2L, black).A second application of agonist then initiates an-other round of cAMP production. In contrast, co-stimulation with rolipram produces two additivephases of intracellular cAMPaccumulation (Fig. 2L,red). Collectively these experiments demonstratethat PDE4 modulates cAMP microdomains sur-rounding anchored and intact PKA holoenzymesProximity ligation assays (PLA) detect endog-

enous protein-protein interactions that occurwithin 40 to 60 nm (15). Unstimulated andagonist-treated human embryonic kidney (HEK)293 cells (HEK293 cells) were fixed, stained withantibodies to RII and C subunits, and subjectedto the PLA amplification protocol before imagingof PLA puncta (fig. S2). Image intensity profilingof PLA puncta showed the distribution of intactPKA holoenzymes (Fig. 2, M to O). The numberof the puncta per cell indicated the extent of RII-Cinteractions under each experimental condition(Fig. 2P). Treatment with isoproterenol did notappreciably change the PLA signal as comparedwith unstimulated control cells (Fig. 2, M, N, andP). In contrast, treatment of cells with isoprote-renol and rolipram reduced the number andintensity of PLA puncta (Fig. 2, O and P). Immu-noblot detection of endogenous RII and C inHEK293 cell lysates confirmed the same trend(fig. S2). Thus, physiological agonists that mobi-

lize cAMP signaling promote very little dissocia-tion of native PKA holoenzymes inside cells.To test whether covalent coupling of RII to the

C subunit would alter PKA action inside cells, wegenerated a construct that encodes RIIa and Cain one polypeptide, creating a nondissociable PKAfusion enzyme designated “R2C2” (Fig. 3A). Toexploit this tool in a simplified genetic background,we used CRISPR-Cas9 editing to disrupt thePRKAR2A,PRKAR2B, andPRKACA genes inU2OShuman osteosarcoma cells [triple-knockout (KO)U2OSR2A/R2B/CA]. Loss of protein expression dueto gene-editing was confirmed by immunoblot(Fig. 3B, lane 2, top three panels). Rescue uponexpression of R2C2was confirmed by immunoblot(Fig. 3C, top, lane 3). Activity profiling using thephospho-PKA substrates antibody confirmed thatisoproterenol-responsive PKA activationwas re-duced to baseline levels in triple-KOU2OSR2A/R2B/CA

cells (fig. S3).Related studies used rapamycin-regulated hetero-

dimerization to “pharmacologically lock” FRBdomain–tagged C subunits (C-FRB) to FK506-

binding protein domain–tagged RII dimers (RII-FKBP) in the context of the AKAP18 complex. Inthis system, the smallmolecule rapamycin bridgesthe tagged proteins to maintain a stable complex(Fig. 3D). Immunoblot analyses of AKAP18 im-mune complexes from cells expressing RII-FKBPand C-FRB revealed that both proteins were asso-ciated after stimulation of cells with isoproterenolin the absence of rapamycin (Fig. 3E, top, lane 2).In keeping with earlier results, administration ofisoproterenol and rolipram together caused supra-physiological accumulation of cAMPand abolishedthis interaction (Fig. 3E, top, lane 3). Conversely,application of rapamycin locks theC-FRB/RII-FKBPsubcomplex together, even when cells are treatedwith isoproterenolplus rolipram(Fig. 3E, top, lane6).We next used A-kinase activity reporter 4

(AKAR4) biosensors that measure PKA activityby changes in FRET to assess how compartmen-talization affects free andpharmacologically lockedPKA activity (Fig. 3F) (16). Cell-based studies intriple-KOU2OS cells confirmed that, within 30 s,cytoplasmic FRET responses to isoproterenol were

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 2 of 6

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 complexof AKAP79 with RII and C subunits of PKA. (B) Stain-free visualization on native gels of intactAKAP79-PKA holoenzyme complex. (C to E) Representative negative-stain EM micrographs ofAKAP79:2RII:2C single particles. Particles were low-pass filtered to 20 Å. (D) Traces outlining theselected particles in (C). (E) Reference-free class averages from RELION 2D alignment and classificationshowing range of particle lengths. (F and G) Schematics illustrating the flexibility and range of motionin AKAP79-anchored PKA holoenzymes. A compact state may favor the phosphorylation of associatedsubstrates (F), whereas an extended conformation may allow PKA to act on multiple distinct localsubstrates (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 afterincubation with increasing cAMP concentrations. Data are presented as means ± SEM. (Inset)Coomassie blue staining of proteins in pull-down experiments.

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robust (Fig. 3G, orange), even after pharmaco-logical locking of this modified PKA holoenzymewith rapamycin (Fig. 3G, blue). Isoproterenoltreatment of cells in the presence of rolipramhas no additional effect (fig. S3). Consequently,

cytoplasmic PKA signaling is sustained whenthe C subunit and the RII dimer are chemicallyconstrained.Further studies with the AKAR4 biosensor and

engineered PKA holoenzymes investigated the

dynamics of cytoplasmic kinase activity. Rapidincreases in cytoplasmic FRET responses wererecorded in U2OSR2A/R2B/CA cells rescued withmurine RIIa and Ca (Fig. 3, H and I, orange). Cellsexpressing the R2C2 fusion protein generated

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 3 of 6

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 inthe presence of phosphodiesterase inhibitors. (D) Schematic depictingRII-CFP and C-YFP intermolecular FRET. (E to H) FRET analysis ofholoenzyme dissociation in HEK293 cells. (E) Time course of representa-tive cells (0 to 400 s) stimulated with isoproterenol. (F) Iso + PDE4inhibitor rolipram. (G) Iso + PDE3 inhibitor milrinone. (H) Amalgamateddata from 25 recordings under each experimental condition. (I) Schematicdepicting ICUE3 cAMP FRET sensor. (J) ICUE3 FRET recordings showcAMP production in response to Iso (black), Iso + rolipram (red), and

forskolin (blue). The number of experiments is indicated. (K and L) FRETanalysis upon two trains of hormonal stimulation. (K) Intermolecular FRETanalysis of holoenzyme dissociation in HEK293 cells upon two trains ofstimulation with isoproterenol (black) and Iso + PDE4 inhibitor rolipram(red). (L) Monitoring cAMP accumulation with ICUE3 FRET sensor inresponse to isoproterenol (black) and Iso + PDE4 inhibitor rolipram(red). Amalgamated data from 25 recordings under each experimentalcondition. (M to O) Proximity ligation assay (PLA) signal-intensityprojections show RII-C interactions in (M) Unstimulated, (N) Iso-stimulated,and (O) Iso- and rolipram-treated HEK293 cells. (P) Quantification ofPLA puncta per cell using Fiji/ImageJ. All data are presented as means ±SEM.

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isoproterenol responses of similar magnitudeand duration (Fig. 3, H and I, green). There wasno detectable FRET response to agonist stimula-tion in the U2OSR2A/R2B/CA deletion backgroundor in cells expressing a catalytically inactivemutant of the RII-C fusion (R2C2K72A) (Fig. 3I,gray, and fig. S3). Thus, covalent coupling of thePKA subunits does not impair cAMP signalingin the cytoplasm and can reconstitute endoge-nous kinase activity.As a further control, we monitored nuclear

PKA activity in both wild-type (WT) U2OS andU2OSR2A/R2B/CA cells usingAKAR4NLS (16).NuclearFRET responses were slow and required supra-physiological accumulation of cAMP after stim-ulation of adenylyl cyclases with 20 mM forskolinin the presence of 3-isobutyl-1-methylxanthine(IBMX) for 30 min (Fig. 3, J and K, and fig. S3).Under these extreme conditions, rescue with mu-rine RIIa and Ca resulted in a gradual increasein FRET over 15 min (Fig. 3, J and K, orange). In

contrast, cells expressing R2C2 showed greatlydiminished nuclear FRET responses (Fig. 3, J andK, green). Together, these results demonstratethat cytoplasmic activation of PKA is a rapid event,whereas nuclear signaling requires persistent andsupraphysiological accumulation of cAMP.Mammalian cells express four regulatory sub-

units and two C subunits of PKA (17). CRISPR/Casgeneeditingof thePRKACBgeneonaU2OSR2A/R2B/CA

deletionbackgroundwas consistently unsuccessful;this suggests that ablationof bothcatalytic genes is alethal event. Yet quadruple-KOU2OSR2A/R2B/CA/CB

cells survived and proliferatedwhen gene editingwas performed in cells stably expressing murineR2C2 (Fig. 3L and fig. S3). Thus, genetically en-gineered cells expressing a single copy of fusedPKA are viable and respond normally to physio-logical stimuli that mobilize the cAMP-signalingpathway. Because intact and active PKA holoen-zymes operate within a radius of 150 to 250 Å oftheir substrates (Fig. 1), compartmentalization

by AKAPs is the critical determinant of PKA-substrate selectivity.An implication of this finding is that kinase

inhibitor drugs operate within the confines ofthese AKAP nanocompartments. As proof of con-cept, we generated an analog-sensitive form ofR2C2 that is exclusively modulated by the cell-permeable kinase inhibitor 1-napthylmethyl-PP1(1-NM-PP1) (Fig. 4A) (18, 19). Engineering thispharmacologically tractable and fused formof PKAwas achieved by replacing the gatekeeper methio-nine in the catalytic moiety of R2C2 with alanine(M120A). In situ characterization of this syn-thetic analog-sensitive kinase in U2OSR2A/R2B/CA

cells was achieved by using an AKAP-derivedPKA-activity reporter (AKAP18RBS-AKAR4) (Fig. 4B).In cells expressing wild-type R2C2, isoproterenol(1 mM) stimulation induced a FRET responsewithin 10 s thatwas refractory to 1-NM-PP1 (Fig. 4C,green). The rate and magnitude of the FRETresponse was attenuated in cells expressing R2C2

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 4 of 6

Fig. 3. CRISPR-Cas9 PKA triple-KO and rescue with an RII-C fusion.(A) Schematic depicting the R2C2 PKA fusion enzyme. (B) Immunoblotsconfirming knockout of PKA subunit proteins: (Top) RIIa, (top middle) RIIb,and (bottom middle) C subunit. (Bottom) Ponceau staining shows equalloading in WT and U2OSR2A/R2B/CA cells. (C) Western blots for PKA Csubunit confirm expression of R2C2 fusion. (D) Schematic of rapamycininduced heterodimerization of PKA subunits. (E) Immunoblots showingthat rapamycin chemically locks the PKA holoenzyme at supraphysiolog-ical concentrations of cAMP. (F) Schematic of AKAR4 PKA activitybiosensor. (G) Cytoplasmic FRET recordings in response to Iso (1 mM)stimulation in U2OSR2A/R2B/CA cells expressing RII-FKBP and C-FRB in the

presence (blue) or absence (orange) of rapamycin (100 nM). (H) Represen-tative cells showing cytoplasmic AKAR4 FRET response upon rescue with(left) RIIa and Ca and (right) R2C2 fusion. (I) Cytoplasmic FRETrecordings intriple-KO U2OSR2A/R2B/CA cells (gray) and cells rescued with R2C2 fusion(green) or WT RIIa and Ca (orange). (J) Montage of cells showing nuclearAKAR4 FRET signals upon rescue with (left) RIIa and Ca or (right) R2C2fusion. (K) Nuclear FRET recordings in U2OSR2A/R2B/CA cells (gray) andcells rescued with R2C2 fusion (green) or WT RIIa and Ca (orange).(L) Proliferation of triple KO U2OSR2A/R2B/CA cells rescued with R2C2 fusion(blue) or two different clones of quadruple KO U2OSR2A/R2B/CA/CB cells rescuedwith R2C2 fusion (red and green).

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M120A, a common property of analog-sensitivekinases (19). However, application of 1-NM-PP1(2 mM) to these cells reduced the FRET signal tobaseline, which indicated strong inhibition of theanalog-sensitive kinase (Fig. 4C, blue). Thus,1-NM-PP1 is an efficient inhibitor of R2C2M120Ain situ.We used this chemically controllable form of

fused PKA to examine phosphorylation of localextra-nuclear substrates. The mitochondrial an-choring protein D-AKAP-1 sequesters PKA atmitochondria to regulate phosphorylation of theproapoptotic protein BAD (Fig. 4D) (20). PKAphosphorylation of BAD Ser155 blocks apoptosisthrough a mechanism involving recruitment of14-3-3 proteins to suppress programmed celldeath (Fig. 4D) (21–23). BAD phosphorylation onSer155 was robustly stimulated by isoproterenolin WT U2OS cells, and this effect was blunted intriple-KO cells (fig. S4). Confocal imaging con-firmed that D-AKAP-1 and BAD are detected atmitochondria (fig. S4), whereas PLAs detectedpuncta that are indicative of an interaction be-tween these two proteins (fig. S4). In triple-KOcells expressing the R2C2 WT and R2C2 M120Amutants, immunoblot analysis confirmed that

these modified kinases effectively phosphorylatedBAD Ser155 after treatment of cells with isopro-terenol (Fig. 4E, top, lanes 1 to 4). Control immu-noblots monitored the expression of the PKAfusion enzymes, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) served as loading con-trols (Fig. 4E,middle and bottom). Application of1-NM-PP1 (2 mM) inhibited the activity of R2C2M120A and blocked phosphorylation of BADSer155

(Fig. 4E, top, lane 6). Normalized BAD pSer155 datafrom three independent experiments is presentedin Fig. 4F. With this tool, we could test whetherlocal activation of PKA is inhibitory towardapoptosis.We induced apoptosis in triple-KO cells express-

ingR2C2M120Aby treatmentwith the chemotoxicagent etoposide (Fig. 4, G and H) (24). Cell deathwas assessed as accumulation of a fluorescentproduct of caspase 3– or 7–mediated proteolysis(Fig. 4G, green) and nuclear condensation ob-served upon staining DNA with 4′,6-diamidino-2-phenylindole (DAPI) (magenta in Fig. 4G).Unstimulated cells exhibit low levels of apoptosis(Fig. 4G, left, and Fig. 4H). Etoposide treatmentpromotes cell death (Fig. 4G, left, middle, andFig. 4H). Stimulation of R2C2 activity with

b-adrenergic receptor agonists (Iso and formo-terol) was protective, as it substantially reducedthe apoptotic index (Fig. 4G, middle right, andFig. 4H). Application of 1-NM-PP1 blocked thisprotective effect (Fig. 4G, right, and Fig. 4H, blue).Thus, fused RII and C subunits can substitute forendogenous PKA in the modulation of local cel-lular events.Contrary to current dogma, our results show

that even in the presence of cAMP, anchoredPKA holoenzymes remain intact and proximal toanchoring sites and substrates. In addition fusionof the kinase moiety to RII supports cAMP sig-naling in diverse cellular contexts. These resultshighlight protein-protein interactions betweenAKAPs and PKA as the primary determent forthe substrate specificity of this broad-spectrumkinase. We also shed light on another interestingfacet of PKA physiology. Of the 545 genes in thehuman kinome, only one other protein kinase,CK2, forms a tetrameric holoenzyme (25). Ourevidence that autoinhibitory and anchoring func-tions are provided by the constitutively associatedRII dimer implies that PKA is more aligned withother members of the kinase superfamily thanoriginally considered.

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Fig. 4. Chemical-geneticand pharmacologicalcontrol of mitochon-drial PKA action.(A) Schematic depictingthe introduction of ananalog-sensitive kinasemutation into the R2C2PKA fusion enzyme. Theactivity of this pharmaco-logically controlled fusionkinase was monitored inU2OSR2A/R2B/CA cells.(B) Representative cellsshowing cytoplasmicAKAR FRET signals atselected time points fromcells expressing (top)R2C2 and (bottom) the1-NM-PP1–sensitiveR2C2 M120A mutant. Theleft image in both panelsshows expression of theFRET reporter. The otherspanels show pseudo-color images of the FRETintensity at one timepoint before stimulation(–45 s), one time pointafter stimulation withisoproterenol (105 s), anda third time point after addition of 1-NM-PP1 (415 s). (C) Time courses ofcytoplasmic AKAR4 FRETresponses to Iso (1 mM, time zero) and 1-NM-PP1(2 mM, 300 s) in PKA triple-KO cells expressing the (green) R2C2 fusionor (blue) the R2C2 M120A mutant. (D) Schematic depicting protectiveeffects of anchored PKA due to phosphorylation of BAD and the resultantrelease of the prosurvival BCL-2 protein. (E) Inhibition of PKA directed BADpSer155 phosphorylation by 1-NM-PP1 in cells expressing R2C2 M120A.Immunoblot detecting (top) BAD pSer155, (middle) R2C2, and (bottom)

GAPDH loading controls. (F) Quantification of BAD pSer155 signal fromimmunoblots from three independent experiments. Data are presented asmeans ± SEM. (G) Fluorescent detection of apoptotic events upon treatmentof cells with the apoptotic agent etoposide, plus and minus b-agonists and1-NM-PP1. Apoptotic cells were detected by (green) activation of caspase and(magenta) condensation of DNA. (H) Quantification of the percentage ofapoptotic cells from (G). The numbers of cells monitored are indicated aboveeach column. Data are presented as means ± SEM.

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Finally, because AKAP79 constrains intact andcatalytically active PKA within 150 to 250 Å, wepropose that the range of anchored PKA action isrestricted to substrates within the immediatevicinity (Fig. 1, F and G). This “signaling island”concept radically changes our viewof howAKAPcomplexes operate and indicates that proteinphosphorylation is muchmore regionally confinedthan previously appreciated (26–29). Anotherprerequisite for this new model is that the loca-tion of these active zones must coincide withmicrodomains where cAMP concentrations fluc-tuate and when substrates are available (10, 30).One exceptionmay be PKA action in the nucleus,where cAMP responsive transcriptional eventsproceed through the phosphorylation of cAMPresponse element–binding protein (CREB) (31, 32).Nonetheless, this intricate molecular architecturehelps explain how common signaling componentsare assembled to elicit distinct, rapid, and transientendocrine responses.

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ACKNOWLEDGMENTS

We thank K. Forbush for technical support, K.F. and N. Pollett formolecular biology and cell culture assistance, all members ofthe Scott Lab for critical discussions, T. Cooke for technicalassistance with FRET microscopy, and M. Milnes for administrativesupport. This work was supported by the following grants fromthe NIH: 5R01DK105542 (J.D.S.), 4P01DK054441 (J.D.S.),1R01GM120553 (D.V.); U.K. Biotechnology and Biological SciencesResearch Council grant BB/L009501/1 (C.E.E.); North WestCancer Research grant CR1037 (P.A.E.); and Core funding fromthe Institute of Integrative Biology, Faculty of Health and LifeSciences, University of Liverpool (C.E.E. and P.A.E.). J.L.E. is therecipient of the Heart and Stroke Foundation of CanadaPostdoctoral Fellowship. F.D.S. and J.D.S. conceived of andsupervised the project. F.D.S., J.L.E., P.J.N., D.P.B., M.V., C.E.E.,P.A.E., and J.D.S. designed the experiments. F.D.S., J.L.E., P.J.N.,D.P.B., M.V., C.E.E., and P.A.E. performed experiments. F.D.S.,J.L.E., P.J.N., D.P.B., M.V., C.E.E., P.A.E., and J.D.S. analyzed thedata. D.V. provided guidance and software for EM experiments.I.S. provided guidance and support for MS experiments withThermo EMR. L.K.L. designed and prepared figures. F.D.S., L.K.L.,and J.D.S. wrote the manuscript.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/356/6344/1288/suppl/DC1Materials and MethodsFigs. S1 to S4Tables S1 to S3References (33–37)

6 September 2016; accepted 24 May 201710.1126/science.aaj1669

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Local protein kinase A action proceeds through intact holoenzymes

Strashnov, Claire E. Eyers, Patrick A. Eyers, Lorene K. Langeberg and John D. ScottF. Donelson Smith, Jessica L. Esseltine, Patrick J. Nygren, David Veesler, Dominic P. Byrne, Matthias Vonderach, Ilya

DOI: 10.1126/science.aaj1669 (6344), 1288-1293.356Science 

, this issue p. 1288Scienceis more precisely targeted within the cell than previously anticipated.localize it in the cell, appears to be restricted to acting within about 200 Å of such anchoring proteins. Thus, PKA activitythat dissociation of the holoenzyme was not necessary for activation. The kinase, which binds anchoring proteins that

closely monitored activation of PKA in cultured human cells and foundet al.complex with the regulatory subunit. Smith (protein kinase A). The textbook view suggests that activation releases the catalytic subunit of the enzyme from its

Many hormone receptors stimulate production of cyclic AMP (adenosine monophosphate), which activates PKAPKA-activation mechanism revised

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