Local Protein Kinase a Action Proceeds Through Intact Holoenzymes F

RESEARCH

SIGNAL TRANSDUCTION observed between mass/charge ratios of 5700 and 6300. These species preferentially contained two, but up to four, molecules of cAMP (fig. S1). When analysis was done at higher concentrations of cAMP Local protein kinase A action (5 mM), the predominant species exhibited cyclic nucleotide occupancy of 4 mol per RII dimer (fig. proceeds through intact holoenzymes S1 and table S3). Notably, the C subunit remained attached under these conditions (fig. S1). Thus, F. Donelson Smith,1 Jessica L. Esseltine,1* Patrick J. Nygren,1 David Veesler,2 we can conclude that a substantial proportion Dominic P. Byrne,3 Matthias Vonderach,3 Ilya Strashnov,4 Claire E. Eyers,3 of the PKA C subunit remained associated with 297–427 Patrick A. Eyers,3 Lorene K. Langeberg,1 John D. Scott1† the AKAP79 -RII dimer when cAMP-binding sites were occupied. Hormones can transmit signals through adenosine 3ʹ,5ʹ-monophosphate (cAMP) to For a more stringent in situ test, we monitored precise intracellular locations. The fidelity of these responses relies on the activation of the integrity of cellular AKAP-PKA complexes in localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory type II (RII) response to ligand activation. AKAP79 or AKAP18g subunits with A-kinase–anchoring proteins (AKAPs) confers location, and catalytic (C) complexes were immunoprecipitated from cell subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that lysates prepared after stimulation of cells with AKAP79 constrains RII-C subassemblies within 150 to 250 angstroms of its targets. the b-adrenergic agonist isoproterenol (Iso, 1 mM). Native mass spectrometry established that these macromolecular assemblies Immunoblot analysis detected equivalent amounts incorporated stoichiometric amounts of cAMP. Chemical-biology– and live cell–imaging of C subunit in samples from cells stimulated with techniques revealed that catalytically active PKA holoenzymes remained intact within the isoproterenol or vehicle control (Fig. 2, A and B, Downloaded from cytoplasm. These findings indicate that the parameters of anchored PKA holoenzyme top, lane 2). Local cAMP flux is controlled through action are much more restricted than originally anticipated. a balance of second-messenger production by adenylyl cyclases and degradation by phospho- diesterases (PDEs) (10). Cells were stimulated with lassical in vitro biochemistry and elegant with the AKAP (Fig. 1F) or in proximity to the isoproterenol in the presence of the general PDE structural studies have led to a commonly peripheral lobes of the extended PKA holoenzyme inhibitor 3-isobutyl-1-methylxanthine (IBMX), the

held view that the active protein kinase A (Fig. 1G). selective PDE3 inhibitor milrinone, or the PDE4- http://science.sciencemag.org/ C (PKA) catalytic (C) subunit dissociates from An implication of our structural model is that, specific inhibitor rolipram (11, 12). The composition the regulatory subunit dimer in the pres- although local cAMP production stimulates ki- of AKAP79-PKA complexes was assessed by im- ence of excess adenosine 3′,5′-monophosphate nase activity, AKAP79:2RII:2C assemblies can munoblot. Intact complexes were detected in con- (cAMP) (1–3). Yet the utility of this rudimentary remain intact. Native mass spectrometry (MS) trol and Iso-treated samples (Fig. 2C, lanes 1 and mechanism inside cells remains unclear (4). Single- allows the measurement of molecular mass and 2). Inclusion of rolipram promoted full dissocia- particle negative-stain electron microscopy (EM) is used to determine the stoichiometry of intact tion of the C subunit (Fig. 2C, lane 3). Likewise, analysis of A-kinase–anchoring protein 79 with protein complexes in the gas phase (7–9). In the application of IBMX induced release of the C sub- PKA (AKAP79:PKA) holoenzyme complexes of presence of cAMP, which copurifies with RII, we unit from AKAP complexes (Fig. 2C, lane 5). In AKAP79, the regulatory II (RII) subunit, and the observed higher-order complexes, including the contrast, inclusion of milrinone had little effect, C subunit (AKAP79:2RII:2C) detected a range of intact 2RII:2C PKA holoenzyme and the pen- indicating that PDE3 is nonfunctional in these

conformationally distinct species (Fig. 1, A to D, tameric AKAP79:2RII:2C assembly (Fig. 1H, fig. S1, AKAP79-PKA microdomains (Fig. 2C, lane 4). Ki- on June 25, 2017 and fig. S1). A set of ~14,000 particles was sub- and tables S1 and S2). From these experiments, nase activation was monitored by immunoblot jected to reference-free two-dimensional (2D) clas- we infer a minimal disruption of the AKAP-PKA detection of phospho-PKA substrates (Fig. 2C, sification using RELION (5). Poorly aligned classes architecture even in the presence of cAMP. bottom panel). Similar findings were obtained were discarded over multiple iterations to reveal To address the relative stability of the pentame- upon analysis of AKAP18g complexes and when an ensemble of three-lobed assemblies in which ric AKAP79:2RII:2C assembly in the presence of prostaglandin E1 (PGE1) or epinephrine was used the peripheral densities are observed at different elevated cAMP, we performed pull-down experi- as an agonist (fig. S2). Treatment with rolipram distances from each other. These range from 150 Å ments with a fragment of the anchoring protein alonedidnotactivatePKAorreleaseCsubunits in the compact configuration to 250 Å in the ex- AKAP79297–427. Retention of anchored C subunits (Fig.2C,lane6).Thus,nativeproductionofcAMP tended conformation (Fig. 1E). The tandem pe- in the presence of increasing concentrations of in response to physiological effectors of GPCR ripheral lobes represent the C subunit in complex cAMP was assessed by quantification of Coomassie signaling appears not to promote C subunit release with the cAMP-binding domains of RII (6), whereas blue–stained protein (Fig. 1I and inset). At physi- from anchored PKA holoenzymes. the central density represents the AKAP-RII di- ological concentrations of cAMP (1 to 2 mM) most Förster resonance energy transfer (FRET) was mer interface (Fig. 1E). Thus, AKAP79 constrains of the C subunit (~70 to 80%) remained associated used to investigate PKA holoenzyme composition each subassembly of regulatory and C subunits with the AKAP79297–427-RII complex (Fig. 1I and in real time (13). Initially, we used RII conjugated to within 250 Å of substrates. We surmise that inset). Substantial release of the C subunit was to cyan fluorescent protein (RII-CFP) and the C the restricted movement of the anchored C sub- only evident at supraphysiological levels of cAMP subunit conjugated to yellow fluorescent pro- unit in this configuration augments phosphoryl- (Fig. 1I; 10 to 90 mM), and no change in binding tein (C-YFP) as intermolecular FRET probes to ation of local substrates that are either interacting of RII to AKAP79297–427 was observed. In control monitor the integrity of the PKA holoenzyme af- experiments, 5′-AMP (100 mM), a degradation ter agonist stimulation of cells (Fig. 2D). Isopro- product of cAMP, did not alter anchored holo- terenol promoted minimal change in the CFP/ 1Howard Hughes Medical Institute, Department of enzyme composition (Fig. 1I). Thus, physiological YFP FRET ratio over time courses of 400 s (Fig. Pharmacology, University of Washington, Seattle, WA 98195, amounts of cAMP promote minimal release of 2, E and H, black), consistent with negligible USA. 2Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. 3Department of Biochemistry, the C subunit from the anchored holoenzyme dissociation of the PKA holoenzyme. In contrast, Institute of Integrative Biology, University of Liverpool, in vitro. preincubation of cells with rolipram before stim- Liverpool L69 7ZB, UK. 4School of Chemistry, The University Additional studies used high-resolution native ulation with b-adrenergic agonist triggered a of Manchester, Manchester M13 9PL, UK. MS to evaluate cAMP occupancy in PKA holo- pronounced and time-dependent reduction in *Present address: Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada. enzyme complexes. At basal cAMP concentrations, the FRET ratio (Fig. 2, F and H, red). Thus, dis- †Corresponding author. Email: [email protected] multiple charge states of 2RII:C subcomplexes were sociation of the PKA holoenzyme only occurred

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 1of6 RESEARCH | REPORT if cAMP accumulated to supraphysiological con- lize cAMP signaling promote very little dissocia- binding protein domain–tagged RII dimers (RII- centrations. Pretreatment of cells with the PDE3 tion of native PKA holoenzymes inside cells. FKBP) in the context of the AKAP18 complex. In inhibitor milrinone had little effect on the FRET To test whether covalent coupling of RII to the this system, the small molecule rapamycin bridges response(Fig.2,GandH,gray).ComparableFRET C subunit would alter PKA action inside cells, we the tagged proteins to maintain a stable complex recordings were obtained when PGE1 was used generated a construct that encodes RIIa and Ca (Fig.3D).ImmunoblotanalysesofAKAP18im- as the agonist (fig. S2). in one polypeptide, creating a nondissociable PKA mune complexes from cells expressing RII-FKBP The ICUE3 FRET biosensor detects agonist- fusion enzyme designated “R2C2” (Fig. 3A). To and C-FRB revealed that both proteins were asso- responsive accumulation of cAMP (Fig. 2I) (14). exploit this tool in a simplified genetic background, ciated after stimulation of cells with isoproterenol Stimulation of cells with isoproterenol or PGE1 we used CRISPR-Cas9 editing to disrupt the intheabsenceofrapamycin(Fig.3E,top,lane2). promoted transient increases in the FRET signal PRKAR2A, PRKAR2B,andPRKACA genes in U2OS In keeping with earlier results, administration of (Fig. 2J, black, and fig. S2). This response was human osteosarcoma cells [triple-knockout (KO) isoproterenol and rolipram together caused supra- enhanced by addition of rolipram (Fig. 2J, red, U2OSR2A/R2B/CA]. Loss of protein expression due physiological accumulation of cAMP and abolished and fig. S2). However, the highest amounts of to gene-editing was confirmed by immunoblot this interaction (Fig. 3E, top, lane 3). Conversely, cAMP production (15 times physiological levels) (Fig. 3B, lane 2, top three panels). Rescue upon application of rapamycin locks the C-FRB/RII-FKBP were recorded upon application of forskolin, a expression of R2C2 was confirmed by immunoblot subcomplex together, even when cells are treated direct activator of adenylyl cyclases and a com- (Fig. 3C, top, lane 3). Activity profiling using the with isoproterenol plus rolipram(Fig.3E,top,lane6). mon tool in cAMP research (Fig. 2J, blue). Thus, phospho-PKA substrates antibody confirmed that We next used A-kinase activity reporter 4 forskolin sustains supraphysiological accumula- isoproterenol-responsive PKA activation was re- (AKAR4) biosensors that measure PKA activity tion of cAMP to concentrations far above those duced to baseline levels in triple-KO U2OSR2A/R2B/CA bychangesinFRETtoassesshowcompartmen- induced by b-agonists. cells (fig. S3). talization affects free and pharmacologically locked We investigated the effect of recurrent stimu- Related studies used rapamycin-regulated hetero- PKA activity (Fig. 3F) (16). Cell-based studies in lation of cAMP synthesis on PKA holoenzyme dimerization to “pharmacologically lock” FRB triple-KO U2OS cells confirmed that, within 30 s, Downloaded from integrity with the same biosensors. After an ini- domain–tagged C subunits (C-FRB) to FK506- cytoplasmic FRET responses to isoproterenol were tial pulse of isoproterenol, the drug was washed out for 500 s before application of a second stimulus (Fig. 2, K and L). Intermolecular FRET revealed that RII-CFP and C-YFP remained in

proximity over the duration of these experiments http://science.sciencemag.org/ (Fig. 2K, black). As expected, costimulation of cells with isoproterenol and rolipram initiated cycles of holoenzyme dissociation and reformation (Fig. 2K, red). Complementary biochemical experiments revealed that washout of rolipram and isoproterenol promoted reformation of AKAP79-PKA holoenzyme over a similar time course (fig. S2). Using the ICUE3 biosensor, we demonstrated that isoproterenol induces a transient rise in the cellular concentration of cAMP that

approaches baseline during washout (Fig. 2L, black). on June 25, 2017 A second application of agonist then initiates another round of cAMP production. In contrast, costimulation with rolipram produces two additive phases of intracellular cAMP accumulation (Fig. 2L, red). Collectively these experiments demonstrate that PDE4 modulates cAMP microdomains sur- rounding anchored and intact PKA holoenzymes Proximity ligation assays (PLA) detect endogenous protein-protein interactions that occur within 40 to 60 nm (15). Unstimulated and agonist-treated human embryonic kidney (HEK) 293 cells (HEK293 cells) were fixed, stained with antibodies to RII and C subunits, and subjected to the PLA amplification protocol before imaging of PLA puncta (fig. S2). Image intensity profiling Fig. 1. AKAP-PKA holoenzyme assemblies remain intact upon cAMP stimulation. (A) SDS– of PLA puncta showed the distribution of intact polyacrylamide gel electrophoresis (SDS-PAGE) and stain-free visualization of a purified complex PKA holoenzymes (Fig. 2, M to O). The number of AKAP79 with RII and C subunits of PKA. (B) Stain-free visualization on native gels of intact of the puncta per cell indicated the extent of RII-C AKAP79-PKA holoenzyme complex. (C to E) Representative negative-stain EM micrographs of interactions under each experimental condition AKAP79:2RII:2C single particles. Particles were low-pass filtered to 20 Å. (D) Traces outlining the (Fig. 2P). Treatment with isoproterenol did not selected particles in (C). (E) Reference-free class averages from RELION 2D alignment and classification appreciablychangethePLAsignalascompared showing range of particle lengths. (F and G) Schematics illustrating the flexibility and range of motion with unstimulated control cells (Fig. 2, M, N, and in AKAP79-anchored PKA holoenzymes. A compact state may favor the phosphorylation of associated P). In contrast, treatment of cells with isoprote- substrates (F), whereas an extended conformation may allow PKA to act on multiple distinct local renol and rolipram reduced the number and substrates (G). (H) Zero-charge state native nanoelectrospray ionization mass spectrum of RII+C+AKAP intensity of PLA puncta (Fig. 2, O and P). Immu- (297–427) complexes. The stoichiometry of AKAP79(297–427) (grey), RII (green), and C subunit noblot detection of endogenous RII and C in (orange) is indicated. (I) Percentage of C subunit bound to AKAP79(297–427)–PKA complexes after HEK293 cell lysates confirmed the same trend incubation with increasing cAMP concentrations. Data are presented as means ± SEM. (Inset) (fig. S2). Thus, physiological agonists that mobi- Coomassie blue staining of proteins in pull-down experiments.

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 2of6 RESEARCH | REPORT robust (Fig. 3G, orange), even after pharmaco- cytoplasmic PKA signaling is sustained when dynamics of cytoplasmic kinase activity. Rapid logical locking of this modified PKA holoenzyme the C subunit and the RII dimer are chemically increases in cytoplasmic FRET responses were with rapamycin (Fig. 3G, blue). Isoproterenol constrained. recorded in U2OSR2A/R2B/CA cells rescued with treatment of cells in the presence of rolipram Further studies with the AKAR4 biosensor and murine RIIa and Ca (Fig.3,HandI,orange).Cells has no additional effect (fig. S3). Consequently, engineered PKA holoenzymes investigated the expressing the R2C2 fusion protein generated Downloaded from http://science.sciencemag.org/

on June 25, 2017

Fig. 2. Anchored PKA holoenzyme dynamics upon ligand stimulation. forskolin (blue). The number of experiments is indicated. (K and L) FRET Western blot analysis of PKA C and RII subunits in (A) AKAP79 or analysis upon two trains of hormonal stimulation. (K) Intermolecular FRET (B) AKAP18g immunoprecipitates (IPs) after isoproterenol stimulation analysis of holoenzyme dissociation in HEK293 cells upon two trains of (Iso). (C) IP and Western blot analysis of AKAP79 complexes isolated in stimulation with isoproterenol (black) and Iso + PDE4 inhibitor rolipram the presence of phosphodiesterase inhibitors. (D) Schematic depicting (red). (L) Monitoring cAMP accumulation with ICUE3 FRET sensor in RII-CFP and C-YFP intermolecular FRET. (E to H) FRET analysis of response to isoproterenol (black) and Iso + PDE4 inhibitor rolipram holoenzyme dissociation in HEK293 cells. (E) Time course of representa- (red). Amalgamated data from 25 recordings under each experimental tive cells (0 to 400 s) stimulated with isoproterenol. (F) Iso + PDE4 condition. (M to O) Proximity ligation assay (PLA) signal-intensity inhibitor rolipram. (G) Iso + PDE3 inhibitor milrinone. (H) Amalgamated projections show RII-C interactions in (M) Unstimulated, (N) Iso-stimulated, data from 25 recordings under each experimental condition. (I) Schematic and (O) Iso- and rolipram-treated HEK293 cells. (P) Quantification of depicting ICUE3 cAMP FRET sensor. (J) ICUE3 FRET recordings show PLA puncta per cell using Fiji/ImageJ. All data are presented as means ± cAMP production in response to Iso (black), Iso + rolipram (red), and SEM.

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 3of6 RESEARCH | REPORT isoproterenol responses of similar magnitude contrast, cells expressing R2C2 showed greatly by AKAPs is the critical determinant of PKA- and duration (Fig. 3, H and I, green). There was diminished nuclear FRET responses (Fig. 3, J and substrate selectivity. no detectable FRET response to agonist stimula- K, green). Together, these results demonstrate An implication of this finding is that kinase tion in the U2OSR2A/R2B/CA deletion background that cytoplasmic activation of PKA is a rapid event, inhibitor drugs operate within the confines of or in cells expressing a catalytically inactive whereas nuclear signaling requires persistent and these AKAP nanocompartments. As proof of con- mutant of the RII-C fusion (R2C2K72A) (Fig. 3I, supraphysiological accumulation of cAMP. cept, we generated an analog-sensitive form of gray, and fig. S3). Thus, covalent coupling of the Mammalian cells express four regulatory sub- R2C2 that is exclusively modulated by the cell- PKA subunits does not impair cAMP signaling units and two C subunits of PKA (17). CRISPR/Cas permeable kinase inhibitor 1-napthylmethyl-PP1 in the cytoplasm and can reconstitute endoge- gene editing of the PRKACB gene on a U2OSR2A/R2B/CA (1-NM-PP1)(Fig.4A)(18, 19). Engineering this nous kinase activity. deletion background was consistently unsuccessful; pharmacologically tractable and fused form of PKA As a further control, we monitored nuclear this suggests that ablation of both catalytic genes is a was achieved by replacing the gatekeeper methio- PKA activity in both wild-type (WT) U2OS and lethal event. Yet quadruple-KO U2OSR2A/R2B/CA/CB nine in the catalytic moiety of R2C2 with alanine U2OSR2A/R2B/CA cells using AKAR4NLS (16). Nuclear cells survived and proliferated when gene editing (M120A). In situ characterization of this syn- FRET responses were slow and required supra- was performed in cells stably expressing murine thetic analog-sensitive kinase in U2OSR2A/R2B/CA physiological accumulation of cAMP after stim- R2C2 (Fig. 3L and fig. S3). Thus, genetically en- cells was achieved by using an AKAP-derived ulation of adenylyl cyclases with 20 mM forskolin gineered cells expressing a single copy of fused PKA-activity reporter (AKAP18RBS-AKAR4) (Fig. 4B). in the presence of 3-isobutyl-1-methylxanthine PKA are viable and respond normally to physio- In cells expressing wild-type R2C2, isoproterenol (IBMX) for 30 min (Fig. 3, J and K, and fig. S3). logical stimuli that mobilize the cAMP-signaling (1 mM) stimulation induced a FRET response Under these extreme conditions, rescue with mu- pathway. Because intact and active PKA holoen- within 10 s that was refractory to 1-NM-PP1 (Fig. 4C, rine RIIa and Ca resulted in a gradual increase zymes operate within a radius of 150 to 250 Å of green). The rate and magnitude of the FRET in FRET over 15 min (Fig. 3, J and K, orange). In their substrates (Fig. 1), compartmentalization response was attenuated in cells expressing R2C2 Downloaded from http://science.sciencemag.org/

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Fig. 3. CRISPR-Cas9 PKA triple-KO and rescue with an RII-C fusion. presence (blue) or absence (orange) of rapamycin (100 nM). (H)Represen- (A) Schematic depicting the R2C2 PKA fusion enzyme. (B) Immunoblots tative cells showing cytoplasmic AKAR4 FRET response upon rescue with confirming knockout of PKA subunit proteins: (Top) RIIa, (top middle) RIIb, (left) RIIa and Ca and (right) R2C2 fusion. (I) Cytoplasmic FRET recordings in and (bottom middle) C subunit. (Bottom) Ponceau staining shows equal triple-KO U2OSR2A/R2B/CA cells (gray) and cells rescued with R2C2 fusion loading in WT and U2OSR2A/R2B/CA cells. (C) Western blots for PKA C (green) or WT RIIa and Ca (orange). (J) Montage of cells showing nuclear subunit confirm expression of R2C2 fusion. (D) Schematic of rapamycin AKAR4 FRET signals upon rescue with (left) RIIa and Ca or (right) R2C2 induced heterodimerization of PKA subunits. (E) Immunoblots showing fusion. (K) Nuclear FRET recordings in U2OSR2A/R2B/CA cells (gray) and that rapamycin chemically locks the PKA holoenzyme at supraphysiolog- cells rescued with R2C2 fusion (green) or WT RIIa and Ca (orange). ical concentrations of cAMP. (F) Schematic of AKAR4 PKA activity (L) Proliferation of triple KO U2OSR2A/R2B/CA cells rescued with R2C2 fusion biosensor. (G) Cytoplasmic FRET recordings in response to Iso (1 mM) (blue) or two different clones of quadruple KO U2OSR2A/R2B/CA/CB cells rescued stimulation in U2OSR2A/R2B/CA cells expressing RII-FKBP and C-FRB in the with R2C2 fusion (red and green).

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 4of6 RESEARCH | REPORT

M120A, a common property of analog-sensitive these modified kinases effectively phosphorylated b-adrenergic receptor agonists (Iso and formo- kinases (19). However, application of 1-NM-PP1 BAD Ser155 after treatment of cells with isopro- terol) was protective, as it substantially reduced (2 mM) to these cells reduced the FRET signal to terenol(Fig.4E,top,lanes1to4).Controlimmu- the apoptotic index (Fig. 4G, middle right, and baseline, which indicated strong inhibition of the noblots monitored the expression of the PKA Fig. 4H). Application of 1-NM-PP1 blocked this analog-sensitive kinase (Fig. 4C, blue). Thus, fusion enzymes, and glyceraldehyde-3-phosphate protective effect (Fig. 4G, right, and Fig. 4H, blue). 1-NM-PP1 is an efficient inhibitor of R2C2 M120A dehydrogenase (GAPDH) served as loading con- Thus, fused RII and C subunits can substitute for in situ. trols (Fig. 4E, middle and bottom). Application of endogenous PKA in the modulation of local cel- We used this chemically controllable form of 1-NM-PP1 (2 mM) inhibited the activity of R2C2 lular events. fused PKA to examine phosphorylation of local M120A and blocked phosphorylation of BAD Ser155 Contrary to current dogma, our results show extra-nuclear substrates.Themitochondrialan- (Fig.4E,top,lane6).NormalizedBADpSer155 data that even in the presence of cAMP, anchored choring protein D-AKAP-1 sequesters PKA at from three independent experiments is presented PKA holoenzymes remain intact and proximal to mitochondria to regulate phosphorylation of the in Fig. 4F. With this tool, we could test whether anchoring sites and substrates. In addition fusion proapoptotic protein BAD (Fig. 4D) (20). PKA local activation of PKA is inhibitory toward ofthekinasemoietytoRIIsupportscAMPsig- phosphorylation of BAD Ser155 blocks apoptosis apoptosis. naling in diverse cellular contexts. These results through a mechanism involving recruitment of We induced apoptosis in triple-KO cells express- highlight protein-protein interactions between 14-3-3 proteins to suppress programmed cell ing R2C2 M120A by treatment with the chemotoxic AKAPs and PKA as the primary determent for death (Fig. 4D) (21–23). BAD phosphorylation on agent etoposide (Fig. 4, G and H) (24). Cell death the substrate specificity of this broad-spectrum Ser155 was robustly stimulated by isoproterenol was assessed as accumulation of a fluorescent kinase. We also shed light on another interesting in WT U2OS cells, and this effect was blunted in product of caspase 3– or 7–mediated proteolysis facetofPKAphysiology.Ofthe545genesinthe triple-KO cells (fig. S4). Confocal imaging con- (Fig. 4G, green) and nuclear condensation ob- human kinome, only one other protein kinase, firmed that D-AKAP-1 and BAD are detected at served upon staining DNA with 4′,6-diamidino- CK2, forms a tetrameric holoenzyme (25). Our mitochondria (fig. S4), whereas PLAs detected 2-phenylindole (DAPI) (magenta in Fig. 4G). evidence that autoinhibitory and anchoring func- Downloaded from puncta that are indicativeofaninteractionbe- Unstimulated cells exhibit low levels of apoptosis tions are provided by the constitutively associated tween these two proteins (fig. S4). In triple-KO (Fig. 4G, left, and Fig. 4H). Etoposide treatment RII dimer implies that PKA is more aligned with cells expressing the R2C2 WT and R2C2 M120A promotes cell death (Fig. 4G, left, middle, and other members of the kinase superfamily than mutants, immunoblot analysis confirmed that Fig. 4H). Stimulation of R2C2 activity with originally considered. http://science.sciencemag.org/ Fig. 4. Chemical-genetic and pharmacological control of mitochon- drial PKA action. (A) Schematic depicting the introduction of an analog-sensitive kinase mutation into the R2C2 PKA fusion enzyme. The activity of this pharmaco-

logically controlled fusion on June 25, 2017 kinase was monitored in U2OSR2A/R2B/CA cells. (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 pseudo- color 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 GAPDH loading controls. (F) Quantification of BAD pSer155 signal from cytoplasmic AKAR4 FRET responses to Iso (1 mM, time zero) and 1-NM-PP1 immunoblots from three independent experiments. Data are presented as (2 mM, 300 s) in PKA triple-KO cells expressing the (green) R2C2 fusion means ± SEM. (G) Fluorescent detection of apoptotic events upon treatment or (blue) the R2C2 M120A mutant. (D) Schematic depicting protective of cells with the apoptotic agent etoposide, plus and minus b-agonists and effects of anchored PKA due to phosphorylation of BAD and the resultant 1-NM-PP1. Apoptotic cells were detected by (green) activation of caspase and release of the prosurvival BCL-2 protein. (E) Inhibition of PKA directed BAD (magenta) condensation of DNA. (H) Quantification of the percentage of pSer155 phosphorylation by 1-NM-PP1 in cells expressing R2C2 M120A. apoptotic cells from (G). The numbers of cells monitored are indicated above Immunoblot detecting (top) BAD pSer155, (middle) R2C2, and (bottom) each column. Data are presented as means ± SEM.

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 5of6 RESEARCH | REPORT

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on June 25, 2017

Smith et al., Science 356, 1288–1293 (2017) 23 June 2017 6of6 Local protein kinase A action proceeds through intact holoenzymes F. Donelson Smith, Jessica L. Esseltine, Patrick J. Nygren, David Veesler, Dominic P. Byrne, Matthias Vonderach, Ilya Strashnov, Claire E. Eyers, Patrick A. Eyers, Lorene K. Langeberg and John D. Scott

Science 356 (6344), 1288-1293. DOI: 10.1126/science.aaj1669

PKA-activation mechanism revised Many hormone receptors stimulate production of cyclic AMP (adenosine monophosphate), which activates PKA (protein kinase A). The textbook view suggests that activation releases the catalytic subunit of the enzyme from its complex with the regulatory subunit. Smith et al. closely monitored activation of PKA in cultured human cells and found that dissociation of the holoenzyme was not necessary for activation. The kinase, which binds anchoring proteins that Downloaded from localize it in the cell, appears to be restricted to acting within about 200 Å of such anchoring proteins. Thus, PKA activity is more precisely targeted within the cell than previously anticipated. Science, this issue p. 1288 http://science.sciencemag.org/

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