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Benchmark A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated (AMPK)

Yan Yan 1,2,3,*, Xin Gu 1,*, H. Eric Xu 1,2 and Karsten Melcher 1,*

1 Laboratory of Structural Sciences and Laboratory of Structural and Biochemistry, Center of and Biology, Van Andel Research Institute, 333 Bostwick Avenue Northeast, Grand Rapids, MI 49503, USA; [email protected] 2 VARI-SIMM Center, Center for Structure and Function of Drug Targets, The CAS Key Laboratory of Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China 3 University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China * Correspondence: [email protected] (Y.Y.); [email protected] (X.G.); [email protected] (K.M.); Tel.: +1-616-234-5699 (K.M.)

Received: 15 August 2017; Accepted: 6 October 2017; Published: 13 October 2017

Abstract: While many methods exist to quantitatively determine activities, 32P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and -absorbing small molecules, and easy quantitation. Here, we demonstrate that the interaction between the yeast Rad53 Forkhead-associated (FHA) domain and a peptide optimized for by AMP-Activated Protein Kinase (AMPK), which has previously been exploited for the generation of intracellular phosphorylation , can serve as a readout for a highly sensitive two-step AMPK AlphaScreen kinase assay with exceptional signal-to-noise ratio.

Keywords: AMP-activated protein kinase (AMPK); Forkhead-associated (FHA) domain; kinase assay; AlphaScreen

1. Introduction AMP-Activated Protein Kinase (AMPK) is a three-subunit protein kinase that functions as central cellular energy and regulator of energy homeostasis in [1–4]. AMPK detects cellular energy states as ratios of AMP, ADP, and ATP (adenylate energy charge [5] ([ATP]+0.5x[ADP])/([ATP]+[ADP]+[AMP])) [6] by competitive binding of all three adenine nucleotides to three separate sites in its γ-subunit [6–8]. Energy stress, i.e., high ratios of AMP, and ADP, to ATP, strongly activate the AMPK kinase activity by multiple mechanisms [9–13], resulting in the phosphorylation of numerous metabolic and regulatory in the cell. The net effect of these phosphorylation events is a cellular reprogramming to activate ATP-generating metabolic pathways, such as glucose and fatty acid mobilization, uptake, and catabolism, and the inhibition of ATP-consuming programs, such as the synthesis of macromolecular building blocks, growth, and proliferation [1,2,4]. AMPK activation is associated with many health benefits, making AMPK a promising target for the treatment of metabolic disorders, including diabetes, obesity, and cancer [3,14,15]. Consequently, development of therapeutic AMPK activity modulators is pursued by many pharmaceutical companies. Direct AMPK activation is determined by various kinase assays, including relatively low throughput radioactive [16,17] and HPLC-based [18] kinase assays, as well as high throughput fluorescence-based assays [4,7,19], which have relatively small signal-to-noise ratios and suffer from fluorescence interference by a substantial fraction of screening compounds. Here, we present a sensitive two-step

Methods and Protoc. 2018, 1, 3; doi:10.3390/mps1010003 www.mdpi.com/journal/mps Methods and Protoc. 2018, 1, 3 2 of 10 kinase assay that is amenable to high throughput screening, has an exceptional (ca. 1000-fold) signal-to-noise ratio, and is resistant to most compound interference. This assay is based on the highly specific, phosphorylation-dependent interaction between a Forkhead-associated (FHA) domain, and an AMPK substrate peptide in an AlphaScreen assay. FHA domains are ~75 stable domains that bind Ser/Thr-phosphorylated peptides [20–22], a binding reaction that has recently been exploited for the generation of intracellular phosphorylation sensors for several protein , including AKT [23], (PKA) [24], ataxia-telangiectasia mutated (ATM) kinase [25], and AMPK [26]. By separating AMPK substrate phosphorylation reactions in the absence or presence of AMPK activity modulators from a subsequent AlphaScreen phospho-substrate/FHA interaction assay, the assay becomes highly robust against compound interference and allows for the determination of absolute activity at substrate concentration at or above Michaelis constant (Km). Given the prominent roles of in signal and of protein kinases as drug targets, this assay can also be adapted to determine the activities of other kinases.

2. Materials and Methods

2.1. Protein Expression and Purification All of the expression constructs were confirmed by DNA sequencing. All of the proteins were expressed from the tricistronic Escherichia coli expression vectors described previously [27,28] H6-α1(13-550)-β1(68-270; S108D)-γ1-AMPK, maltose-binding protein (MBP)-α1(13-550)-β1(68-270; S108D)-γ1(24-327)-AMPK, and MBP-α1(11-550)-β2-γ1-AMPK expression plasmids were transformed into E. coli BL21 (DE3) (AMPKα1(13-550): NCBI/GenBank: AAH37303.1; AMPKβ1(68-270): UniProt ID Q9Y478, NCBI: NP_006244.2; AMPKβ2: UniProt ID O43741; AMPKγ1: NCBI: NP_002724.1. His6-AMPK and MBP-AMPK exhibit identical fold activation by AMP and pharmacological activators, but MBP-AMPK has slightly higher total kinase activity. Cells were grown in LB medium (1% w/v ◦ tryptone, 0.5% w/v yeast extract, 1% w/v NaCl) to an OD600 of ~1 at 28 C and induced with 100 µM isopropyl-β-D-thio-galactopyranoside (IPTG) at 16 ◦C overnight. Cell pellets were resuspended in His/MBP buffer A (His buffer A: 25 mM Tris, pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol, 5 mM β-mercaptoethanol; MBP buffer A: 25 mM Tris, pH 8.0, 300 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic_acid (EDTA), 10% glycerol, 2 mM dithiothreitol (DTT), and lysed by French Press with pressure set to 900 Pa. Lysates were cleared by centrifugation for 30 min at 20,000× g at 4 ◦C, passed over a 10 mL HisTrap HP column/MBPTrap HP column (GE Healthcare), and eluted with His/MBP buffer B (His buffer B: 25 mM Tris, pH 8.0, 300 mM NaCl, 500 mM imidazole, 10% glycerol, 5 mM β-mercaptoethanol; MBP buffer B: 25 mM Tris, pH 8.0, 300 mM NaCl, 40 mM maltose, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, 2 mM DTT). The eluted AMPK was further purified by size-exclusion chromatography through a HiLoad 26/60 Superdex 200 column (GE Healthcare) in 25 mM Tris, pH 8.0, 300 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, 2 mM DTT. Phosphorylated AMPK was generated by incubation with 0.02-fold molar ratio of CaMKKβ in 0.2 mM AMP, 0.2 mM ATP, 2 mM CaCl2, 10 mM DTT, and 1 µM at room temperature overnight (16 h). The phosphorylated AMPK was further purified by size-exclusion chromatography through a HiLoad 26/60 Superdex 200 column (GE Healthcare) in 25 mM Tris, pH 8.0, 300 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, 2 mM DTT. The His6GST-FHA purification protocol is the same as for His-tagged AMPK. The FHA domain corresponds to Rad53(22-162) (NCBI Reference Sequence: NP_015172.1). The protein eluted from the gel filtration column at a volume corresponding to the size of a monomeric complex at a purity ≥95% as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure S1).

2.2. AMPK AlphaScreen Kinase Assay 10 nM AMPK were incubated with 50 µM b-ASP (biotinylated AMPK substrate peptide: biotin-GSTKMRRVATLVDLGYKK; synthesized by Peptide 2.0 Inc., Chantilly, VA, USA) and 100 µM Methods and Protoc. 2018, 1, 3 3 of 10

ATP in kinase buffer (25 mM Tris, pH 8.0, 300 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, 2 mM DTT) in a microcentrifuge tube in a total volume of 10 µL for 20 min at room temperature (non-phosphorylated AMPK) or on ice (phosphorylated AMPK). The reaction was terminated by a two-step 1000-fold dilution by adding 490 µL kinase buffer (50-fold dilution), of which 7.5 µL were added to a total volume of 150 µL in AlphaScreen buffer (50 mM MOPS, pH7.4, 50 mM NaF, 50 mM CHAPS, and 0.1 mg/mL bovine serum albumin) containing 50 nM His6GST-FHA, 5 µg/mL AlphaScreen Streptavidin-coated Donor beads and 5 µg/mL Nickel-chelate Acceptor beads (PerkinElmer, Waltham, MA, USA). AlphaScreen reactions were incubated for 1.5 h in the dark at room temperature. Donor and acceptor beads are brought into proximity by the interaction between b-pASP and His6GST-FHA. When excited by a laser beam of 680 nm, the photosensitizer in the donor beads converts ambient oxygen into short-lived singlet oxygen that can transfer energy to the thioxene derivatives in the acceptor beads, releasing photons of 520–620 nm as the binding signal that can be measured in an Envision (PerkinElmer) plate reader. For absolute activity determinations, nM p-ASP in the AlphaScreen reaction was determined by interpolation from the calibration curve using the “Interpolate unknowns function by non-linear regression” of GraphPad Prism. Since the AlphaScreen reaction was performed with 1/1000th of the , total first step kinase reaction (1000-fold dilution), nM p-ASP in the AlphaScreen reaction corresponds to µM substrate conversion in the first step kinase reaction. The kinase reaction contains 0.01 µM AMPK and proceed for 20 min, absolute activities are therefore 1 µM p-ASP per 0.01 µM AMPK per 20 min for every 1 nM p-ASP in the AlphaScreen reaction, i.e., each nM p-ASP in the AlphaScreen reaction corresponds to 100 µmol substrate conversion/(µM AMPK × 20 min) = 5 µmol substrate conversion × (µmole AMPK)−1 × min−1 (which corresponds to 0.6 nmol mg−1 AMPK min−1 for H6-AMPK (120 kDa)).

2.3. AMPK [32P]-γATP Kinase Assay For the radioactive kinase assay, 10 nM AMPK were incubated with 50 µM b-ASP, 2 mM DTT, 32 and 0.25 µL[ P]-γATP per 15 µL reaction in kinase buffer (25 mM Tris, pH 7.4, 12 mM MgCl2, 1 mM Na3VO4, 5 mM NaF) for 30 min at room temperature. Reactions were terminated by addition of 0.5 volumes of 7.5 M guanidine hydrochloride solution in water and reactions were spotted on a SAM 2® Biotin Capture Membrane (Promega, Madison, WI, USA). The membrane was washed once for 30 s with 2 M NaCl, 3 times for 2 min with 2 M NaCl, 4 times for 2 min with 2 M NaCl in 1% H3PO4, and two times for 30 s with deionized water to remove unbound reaction components. Membranes were dried at room temperature for 30–60 min, and signals were quantitated by PhosphorImager analysis.

3. Results and Discussion

3.1. The FHA Domain Can Highly Selectively Distinguish Phosphorylated from Non-Phosphorylated AMPK Substrate Peptide AMPKAR is a FRET-based AMPK activity sensor that detects the phosphorylation-dependent intramolecular interaction between an enhanced yellow fluorescent protein (eCFP)-tagged yeast Rad53 FHA domain and an optimized Venus-tagged AMPK substrate peptide in the context of an eCFP-FHA-peptide-Venus fusion protein [26]. To test whether the FHA/phospho-peptide interaction can be utilized in trans (inter-molecularly) for quantitative determination of AMPK kinase activity, we synthesized a biotinylated version of the AMPK substrate peptide (b-ASP; Figure1a) and expressed and purified the FHA domain as His6GST-fusion protein. We then subjected b-ASP and His6GST-FHA in a standard kinase buffer in the presence and absence of non-phosphorylated α1β1γ1-AMPK to a one-step combined kinase and AlphaScreen reaction. In this reaction, AMPK phosphorylates streptavidin donor bead-bound b-ASP to b-pASP, which interacts with Ni acceptor bead-bound His6GST-FHA to bring donor and acceptor beads into close proximity for generation of an amplified, singlet oxygen mediated luminescence signal (Figure1a). Even though non-phosphorylated AMPK Methods and Protoc. 2018, 1, 3 4 of 10 has less than 1% of the activity of phosphorylated AMPK [29,30], we observed a strong luminescence signal that was dependent on both interaction partners, b-ASP and His6GST-FHA, as well as on AMPKMethods (Figure Protoc.1b), 2017 suggesting, 1, 3 that the b-ASP/FHA interaction requires phosphorylation4 of of 10 the centralrequirement threonine byusing AMPK. a Wesynthetic further validatedphospho‐threonine the phosphorylation containing requirementb‐pASP peptide using (biotin a synthetic‐ p p phospho-threonineGSTKMRRVApT containingLVDLGYKK) b- andASP tested peptide the luminescence (biotin-GSTKMRRVA interaction signalTLVDLGYKK) of both phosphorylated and tested the luminescenceand non‐ interactionphosphorylated of both side phosphorylated by side. As shown and in non-phosphorylated Figure 1c, b‐pASP interacted peptide strongly side by side. As shownwith the in Figure FHA domain,1c, b- pASP whereas interacted no interaction strongly was with detectable the FHA with domain, the non whereas‐phosphorylated no interaction b‐ASP was detectablepeptide. with the non-phosphorylated b-ASP peptide.

Figure 1. One-step AlphaScreen kinase assay. (a) Schematic presentation of the assay. Incubation Figure 1. One‐step AlphaScreen kinase assay. (a) Schematic presentation of the assay. Incubation of of streptavidin donor bead-bound biotinylated version of the AMPK substrate peptide (b-ASP) streptavidin donor bead‐bound biotinylated version of the AMPK substrate peptide (b‐ASP) with H6‐ with H6-α1(13-550)-β1(68-270; S108D)-γ1-AMPK leads to b-ASP phosphorylation, which in turns α1(13‐550)‐β1(68‐270; S108D)‐γ1‐AMPK leads to b‐ASP phosphorylation, which in turns allows b‐ allowsASP b-ASP to bind to H6 bind‐FHA H6-FHA immobilized immobilized to Ni‐charged to Ni-charged acceptor beads. acceptor This interaction beads. This brings interaction acceptor and brings acceptordonor and beads donor into beads close proximity into close to proximity generate an to amplified, generate singlet an amplified, oxygen‐mediated singlet oxygen-mediated luminescence luminescenceproximity proximitysignal; (b) signal;AMP‐Activated (b) AMP-Activated Protein Kinase Protein (AMPK) Kinase mediates (AMPK) a robust mediates AlphaScreen a robust AlphaScreenluminescence luminescence signal that signal is dependent that is dependenton b‐ASP and on His6 b-ASP‐FHA; and (c) His6-FHA; Non‐phosphorylated (c) Non-phosphorylated b‐ASP does b-ASPnot does interact not with interact the withForkhead the‐associated Forkhead-associated (FHA) domain (FHA) in the domain AlphaScreen in the assay. AlphaScreen Number of assay. Numberreplicates of replicates = 3, error = 3,bars error = standard bars = standarddeviation. deviation.ADP: ADP: diphosphate, adenosine ATP: diphosphate, adenosine ATP: adenosinetriphosphate triphosphate.

3.2. A Two‐Step Assay Allows Determination of Absolute AMPK Kinase Activities 3.2. A Two-StepThe streptavidin Assay Allows donor Determination beads have ofa b Absolute‐ASP binding AMPK capacity Kinase of Activities about 50 nM, a concentration Thethat streptavidinis two orders of donor magnitude beads below have the a b-ASP Km of bindingthe reaction capacity and leads of aboutto substrate 50 nM, exhaustion. a concentration We that istherefore two orders set up of a two magnitude‐step assay below (Figure the 2a) Km to allow of the in reactionthe first step and the leads kinase to reaction substrate to proceed exhaustion. at substrate concentrations (ATP: 100 μM; b‐ASP peptide: 50 μM) above Km (Km ATP: 26–35 μM; We therefore set up a two-step assay (Figure2a) to allow in the first step the kinase reaction to Km of less optimal AMPK substrate peptide SAMS: 27 μM) [4,31], and then dilute the reaction 1000‐

Methods and Protoc. 2018, 1, 3 5 of 10 proceedMethods Protoc. at substrate 2017, 1, 3 concentrations (ATP: 100 µM; b-ASP peptide: 50 µM) above Km (Km5 of 10 ATP: 26–35 µM; Km of less optimal AMPK substrate peptide SAMS: 27 µM) [4,31], and then dilute the fold to achieve a low concentration (50 nM) of total b‐ASP (b‐ASP + b‐pASP) that matches the p reactionstreptavidin 1000-fold donor to bead achieve capacity a low during concentration the subsequent (50 nM) (second of total b-ASPstep) Alphascreen (b-ASP + b- reactionASP) that in matches384 thewell streptavidin plates. Because donor AMPK bead phosphorylates capacity during b the‐ASP subsequent more efficiently (second than step) the Alphascreen commonly used reaction SAMS in 384 wellpeptide plates. substrate, Because it was AMPK important phosphorylates to establish b-ASP conditions more at efficiently which AMPK than does the commonly not cause substrate used SAMS peptideexhaustion. substrate, While itthe was simplest important way to to limit establish b‐ASP conditions phosphorylation at which is by AMPK AMPK does dilution, not cause we found substrate exhaustion.that the AMPK While heterotrimer the simplest becomes way tounstable limit b-ASP and loses phosphorylation catalytic activity is byat concentrations AMPK dilution, ≤100 we pM found that(Figure the AMPK2b). We heterotrimer therefore kept becomes the concentration unstable and of AMPK loses catalytic constant activity at 10 nM at concentrationsand incubated for≤100 20 pM (Figuremin either2b). Weat room therefore temperature kept the (non concentration‐phosphorylated of AMPK AMPK) constant or on at ice 10 nM(phosphorylated and incubated AMPK), for 20 min eitherconditions at room at which temperature AMPK (non-phosphorylateddoes not cause substrate AMPK) exhaustion or on and ice (phosphorylated remains completely AMPK), stable. conditions 1000‐ atfold which dilution AMPK efficiently does not and cause immediately substrate terminates exhaustion the and reaction remains (Figure completely 2c) due stable. to the 1000-fold instability dilution of efficientlyAMPK at 10 and pM immediately concentration terminates and due to the concentrations reaction (Figure of ATP2c) due and to b‐ theASP instability that are about of AMPK two orders at 10 pM concentrationof magnitude below and due Km. to concentrations of ATP and b-ASP that are about two orders of magnitude below Km.

Figure 2. Two-step AlphaScreen kinase assay. (a) Schematic assay presentation. (b) AMPK is inactiveFigure 2. when Two‐step diluted AlphaScreen to ≤0.1 nM. kinase Activities assay. were(a) Schematic determined assay by presentation. standard radioactive (b) AMPK is peptide inactive assay. (whenc) 1000-fold diluted dilution to ≤0.1 nM. terminates Activities the were kinase determined (Step 1) by reaction. standard The radioactive Step 1 reaction peptide assay. was performed (c) 1000‐ in thefold presence dilution ofterminates32P-γATP, the diluted kinase 1000-fold, (Step 1) reaction. and then The either Step directly 1 reaction spotted was on performed streptavidin-coated in the filterpresence paper of or32P‐γ afterATP, a twodiluted hour 1000 additional‐fold, and incubation then either indirectly the diluted spotted kinase on streptavidin buffer. 32‐Pcoated incorporation filter intopaper the or b-ASPafter a two peptide hour wasadditional determined incubation by in PhosphorImager the diluted kinase analysis. buffer. 32P Number incorporation of replicates into the = 3, errorb‐ASP bars peptide = standard was determined deviation; by rt: PhosphorImager room temperature. analysis. Number of replicates = 3, error bars = standard deviation; rt: room temperature. An additional advantage of the two-step protocol is that it allows the performing of the reactionAn additional in the presence advantage of most of the fluorescent two‐step protocol and/or is light-absorbing that it allows the compounds, performing of which the reaction represent ain significant the presence fraction of most of fluorescent molecules and/or in common light‐absorbing drug screening compounds, compound which libraries.represent a To significant test whether AMPK-modulatingfraction of molecules compounds in common interfere drug screening with the compound AlphaScreen libraries. signal, To we test incubated whether a biotinylatedAMPK‐ modulating compounds interfere with the AlphaScreen signal, we incubated a biotinylated Gly6His6 Gly6His6 control peptide (b-Gly6His6; binds simultaneously acceptor and donor beads) with ATP and control peptide (b‐Gly6His6; binds simultaneously acceptor and donor beads) with ATP and

Methods and Protoc. 2018, 1, 3 6 of 10 Methods Protoc. 2017, 1, 3 6 of 10 prominent pharmacological AMPK AMPK activators activators at at concentrations concentrations at at which which they they are are typically used in AMPK kinase kinase assays assays as as well well as as at at 1000 1000-fold‐fold dilution dilution of ofthe the kinase kinase reactions. reactions. While While MT47 MT47-100‐100 [32] and [32] A769662and A769662 [33] [clearly33] clearly interfered interfered with with the theassay assay at 50 at μ 50Mµ Mand and 10 10 μM,µM, each, each, none none of of the the AMPK modulators interfered with the signals in the 1000-fold1000‐fold diluted reactions, as was expected (Figure(Figure3 3).).

Figure 3. Reaction dilution abolishes signal interference by pharmacological AMPK activators. (a) Schematic of the control AlphaScreen reaction; (b) MT47-100 and A769662 interfere with the Figure 3. Reaction dilution abolishes signal interference by pharmacological AMPK activators. (a) luminescence signal at concentrations used in kinase assays. No interference is detectable at 1000-fold Schematic of the control AlphaScreen reaction; (b) MT47‐100 and A769662 interfere with the diluted compound concentrations. Number of replicates = 3, error bars = standard deviation. luminescence signal at concentrations used in kinase assays. No interference is detectable at 1000‐fold diluted compound concentrations. Number of replicates = 3, error bars = standard deviation. 3.3. Assay Normalization 3.3. AssayTo calibrate Normalization the assay for quantitation, we generated Alphascreen dose-response curves using 50 nMTo b-ASP calibrate at defined the assay ratios for of quantitation, non-phosphorylated we generated and phosphorylated Alphascreen dose peptide.‐response As seen curves in Figure using4, p 50we nM detected b‐ASP steep at defined dose-response ratios of curves, non‐phosphorylated allowing for accurate and phosphorylated activity measurements peptide. withinAs seen the in b- FigureASP 4,concentration we detected rangesteep betweendose‐response initial curves, signal increases allowing andfor accurate the beginning activity signal measurements saturation. within Since thisthe brange‐pASP is concentration tight, it is important range between to include initial a calibration signal increases data set on and the the same beginning plate that signal contains saturation. the test Since data. thisIn those range cases is tight, where it testis important data fall outside to include of the a calibration variable range data of set the on calibration the same curve,plate that assay contains conditions the testneed data. to be In adapted those bycases changes where in test incubation data fall time outside or dilution. of the Asvariable a test case range we of generated the calibration dose-response curve, assaycurves conditions for the pharmacological need to be adapted activators by 991changes [34] (Figure in incubation4A) and time A769662 or dilution. [ 33] (Figure As a4 B),test as case well we as generatedfor the physiological dose‐response activator curves AMP for (Figurethe pharmacological4C). Given the activators different protein 991 [34] preparations, (Figure 4A) and EC50 A769662 values [33]and fold(Figure activation 4B), as are well in similaras for rangethe physiological as published activator for radioactive AMP assays(Figure using 4C). theGiven SAMS the peptide different as proteinsubstrate preparations, (991: EC50 =EC50 ~80 values nM, 11-fold and fold activation activation [34 are]; A769662: in similar EC50 range = as ~80 published nM, 2.7-fold for radioactive activation; assaysAMP: EC50using = the ~3 µSAMSM, 3.7-fold peptide activation as substrate for non-phosphorylated (991: EC50 = ~80 nM, AMPK 11‐fold [30 ,35activation]. [34]; A769662: EC50 = ~80 nM, 2.7‐fold activation; AMP: EC50 = ~3 μM, 3.7‐fold activation for non‐phosphorylated AMPK [30,35].

MethodsMethods and Protoc. Protoc. 20172018, 1,, 31 , 3 77 of of 10 10

Calibration curve Photon counts Activity units

A. 991 EC50=29 nM 1000000 500000 10 6.3fold activation -1 800000 400000 8 mg 600000 300000 -1 6

400000 200000 4 Photon Counts Photon Photon Counts Photon 200000 100000 2 nmole min nmole

0 0 0 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 -1 10 0 10 1 10 2 10 3 10 4 10 -1 10 0 10 1 10 2 10 3 10 4 Concentration of p-bioASP (nM) Concentration of 991 (nM) Concentration of 991 (nM)

B. A769662 Photon counts Activity units

800000 12 EC50=149 nM 6.4-fold activation 600000 ‐1

mg 8 ‐1 400000 4 200000 Photon Counts Photon nmole min 0 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 0 -1 10 0 10 1 10 2 10 3 10 4 10 5 Concentration of A769662 (nM) Concentration of A769662 (nM)

C. AMP Photon counts Activity units 400000 5 EC50=6.1 µM

-1 4.2-fold activation 300000 4 mg

-1 3 200000 2 100000

Photon Counts 1 nmole min nmole 0 0 10 -2 10-1 100 10 1 10 2 10 3 10 -2 10 -1 10 0 10 1 10 2 10 3 Concentration of AMP (M) Concentration of A769662 (nM)

Figure 4. Determination of absolute kinase activities of non-phosphorylated H6-α1(13-550)-β1(68-270; S108D)-Figureγ 4.1-AMPK Determination calibration. of absolute (a) 991; kinase (b) A769662; activities (c )of AMP. non‐phosphorylatedN = 3, error bars H6 = SD.‐α1(13‐550)‐β1(68‐270; S108D)‐γ1‐AMPK calibration. (a) 991; (b) A769662; (c) AMP. N = 3, error bars = SD. 3.4. Sensitivity of the Forkhead-associated-AlphaScreen Assay Is Comparable to that of a Radioactive Kinase3.4. Sensitivity Assay of the Forkhead‐associated‐AlphaScreen Assay Is Comparable to That of a Radioactive Kinase Assay Currently, radioactive kinase assays are the gold standard for assay sensitivity. To compare the sensitivitiesCurrently, of the radioactive AMPK FHA-AlphaScreen kinase assays are assay the withgold astandard [32P]-ATP for kinase assay assay,sensitivity. we incubated To compare 10 nM, the 100sensitivities nM, and 1 ofµ gthe low-activity AMPK FHA non-phosphorylated‐AlphaScreen assay MBP- withα1 aβ 1[32γP]1-AMPK‐ATP kinase in the assay, presence we or incubated absence of 10 32nM,P-ATP 100 with nM, b-ASP and 1 substrate μg low‐ foractivity 10 min non on‐ icephosphorylated (to minimize kinaseMBP‐α activity).1β1γ1‐AMPK We captured in the b-ASPpresence from or theabsence radioactive of 32P‐ATP kinase with assay b‐ASP on streptavidin-coatedsubstrate for 10 min on filters ice (to and minimize exposed kinase them toactivity). a PhosphorImager We captured screenb‐ASP either from forthe 4 radioactive h or overnight. kinase b-ASP assay from on thestreptavidin non-radioactive‐coated reactionfilters and was exposed 1000-fold them diluted to a andPhosphorImager analyzed in the screen AlphaScreen either for assay. 4 h or Asovernight. shown inb‐ FigureASP from5, both the thenon AlphaScreen‐radioactive reaction assay and was the1000 radioactive‐fold diluted assay and had analyzed similar in sensitivity the AlphaScreen limits and assay. both As were shown clearly in Figure capable 5, both to detect the AlphaScreen substrate phosphorylationassay and the radioactive in a reaction assay with had 100 similar ng non-phosphorylated sensitivity limits AMPKand both at 4were◦C, conditionsclearly capable under to which detect AMPKsubstrate has phosphorylation very low activity. in While,a reaction the with total 100 PhosphorImager ng non‐phosphorylated counts were AMPK higher at after4 °C, overnightconditions exposure,under which fold AMPK signal changeshas very were low activity. actually While, larger afterthe total 4 h exposurePhosphorImager (shown incounts Figure were5b). higher after overnight exposure, fold signal changes were actually larger after 4 h exposure (shown in Figure 5b).

Methods Protoc. and Protoc. 20172018, 1, 3, 1, 3 8 of 10

(a)(AlphaScreen assay b) Radioactive assay

500000 60000 400000 300000 40000 cpm 40000 20000

Photon Counts Photon 20000 0 0

PK PK MPK M -AM A P-A P- BP BP-AMPK BP-AMPK M M M MB MB M M M MBP AMPK n  nM  1 0 nM 0 1 10 1 100 nM 10

Figure 5. Side-by-side sensitivity comparison of AlphaScreen and radioactive kinase assays. Indicated Figure 5. Side‐by‐side sensitivity comparison of AlphaScreen and radioactive kinase assays. Indicated concentrations of non-phosphorylated MBP-α1(11-550)-β2-γ1-AMPK were incubated with b-ASP for concentrations of non‐phosphorylated MBP‐α1(11‐550)‐β2‐γ1‐AMPK were incubated with b‐ASP for 10 minutes on ice and phosphoryl transfer determined by Two-step AlphaScreen kinase assay (a) or by 10 minutes on ice and phosphoryl transfer determined by Two‐step AlphaScreen kinase assay (a) or PhosphorImager quantitation of the radiolabel transfer (b). N = 3, error bars = SD. by PhosphorImager quantitation of the radiolabel transfer (b). N = 3, error bars = SD. 4. Conclusions 4. Conclusions In this paper, we present a highly sensitive non-radioactive AMPK kinase assay that can be In this paper, we present a highly sensitive non‐radioactive AMPK kinase assay that can be performed for high-throughput screening in 384-well plates. This assay is very affordable as it does not performed for high‐throughput screening in 384‐well plates. This assay is very affordable as it does depend on commercial and/or expensive fluorescent tracers, but rather uses recombinant not depend on commercial antibodies and/or expensive fluorescent tracers, but rather uses FHA protein that can be expressed and purified from E. coli in large quantities with minimal effort. recombinant FHA protein that can be expressed and purified from E. coli in large quantities with A hallmark of this assay is its exceptional signal-to-noise ratio due to the steep response curve, which minimal effort. A hallmark of this assay is its exceptional signal‐to‐noise ratio due to the steep makes it ideal for highly sensitive compound screening. However, the steep response limits linearity response curve, which makes it ideal for highly sensitive compound screening. However, the steep of the assay to a relatively small product concentration range, which makes parallel determination of a response limits linearity of the assay to a relatively small product concentration range, which makes calibration curve essential. Quantitative determination requires that all of points of the dose-response parallel determination of a calibration curve essential. Quantitative determination requires that all of curve fall within the linear range of the calibration curve. Since this can be challenging, together points of the dose‐response curve fall within the linear range of the calibration curve. Since this can with the high sensitivity of the assay, it is best suited for initial high throughput compound screening. be challenging, together with the high sensitivity of the assay, it is best suited for initial high We note that while this assay has been specifically designed for determination of AMPK activity, it can throughput compound screening. We note that while this assay has been specifically designed for in principle be adapted to any protein kinase. determination of AMPK activity, it can in principle be adapted to any protein kinase. Supplementary Materials: The following are available online at www.mdpi.com/2409-9279/1/1/3/s1, Figure S1: SupplementarySize exclusion chromatography Materials: The following and SDS PAGE are available profiles online of purified at www.mdpi.com/link, proteins. Figure S1: Size exclusion chromatography and SDS PAGE profiles of purified proteins. Acknowledgments: This work was supported by the Van Andel Research Institute (H.E.X. and K.M.), Ministry Acknowledgments:of Science and Technology This work (China) was supported grants 2012ZX09301001, by the Van Andel 2012CB910403, Research Institute 2013CB910600, (H.E.X. and K.M.), XDB08020303, Ministry of2013ZX09507001, Science and Technology NSF 91217311 (China) (H.E.X.), grants and National 2012ZX09301001, Institute of Health2012CB910403, grants DK071662 2013CB910600, (H.E.X.) andXDB08020303, GM104212 (K.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views 2013ZX09507001,of the National Institutes NSF 91217311 of Health. (H.E.X.), and National Institute of Health grants DK071662 (H.E.X.) and GM104212 (K.M.). The content is solely the responsibility of the authors and does not necessarily represent the officialAuthor views Contributions: of the NationalY.Y. and Institutes X.G. conducted of Health. the experiments; H.E.X., and K.M. conceived the study and analyzed results; and K.M. wrote the paper with comments from all authors. AuthorConflicts Contributions: of Interest: The Y.Y. authors and X.G. declare conducted no conflict the of experiments; interest. H.E.X., and K.M. conceived the study and analyzed results; and K.M. wrote the paper with comments from all authors.

ConflictsReferences of Interest: The authors declare no conflict of interest.

1. Hardie, D.G.; Ross, F.A.; Hawley, S.A. AMPK: A nutrient and energy sensor that maintains energy References homeostasis. Nat. Rev. Mol. Cell Biol. 2012, 13, 251–262. [CrossRef][PubMed] 1.2. Hardie,Hardie, D.G.; DG, Schaffer, FA Ross, B.E.; and Brunet, SA Hawley, A. AMPK: AMPK: An Energy-Sensing a nutrient and Pathwayenergy sensor with that Multiple maintains Inputs energy and Outputs.homeostasis.Trends Nat Cell Rev Biol. Mol2016 Cell, 26 ,Biol, 190–201. 2012. [ 13CrossRef: 251‐62.][ PubMed] 2. Hardie, DG, BE Schaffer, and A Brunet, AMPK: An Energy‐Sensing Pathway with Multiple Inputs and Outputs. Trends Cell Biol, 2016. 26: 190‐201. 3. Steinberg, GR and BE Kemp, AMPK in Health and Disease. Physiol Rev, 2009. 89: 1025‐78.

Methods and Protoc. 2018, 1, 3 9 of 10

3. Steinberg, G.R.; Kemp, B.E. AMPK in Health and Disease. Phys. Rev. 2009, 89, 1025–1078. [CrossRef] [PubMed] 4. Reichling, L.J.; Riddle, S.M.; Mei, B.; Bruinsma, R.; Goossens, T.A.; Huwiler, K.G.; Maffitt, M.; Newport, A.M.; Qian, X.D.; Ruttimann-Johnson, C.; et al. Homogenous fluorescent assays for characterizing small-molecule activators of AMP-activated protein kinase (AMPK). Curr. Chem. Genom. 2008, 1, 34–42. [CrossRef][PubMed] 5. Atkinson, D.E.; Walton, G.M. conservation in metabolic regulation rat liver citrate cleavage enzyme. J. Biol. Chem. 1967, 242, 3239–3241. [PubMed] 6. Oakhill, J.S.; Steel, R.; Chen, Z.P.; Scott, J.W.; Ling, N.; Tam, S.; Kemp, B.E. AMPK is a direct adenylate charge-regulated protein kinase. Science 2011, 332, 1433–1435. [CrossRef][PubMed] 7. Sinnett, S.E.; Sexton, J.Z.; Brenman, J.E. A High Throughput Assay for Discovery of Small Molecules that Bind AMP-activated Protein Kinase (AMPK). Curr. Chem. Genom. Transl. Med. 2013, 7, 30–38. [CrossRef] [PubMed] 8. Gowans, G.J.; Hawley, S.A.; Ross, F.A.; Hardie, D.G. AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation. Cell Metab. 2013, 18, 556–566. [CrossRef][PubMed] 9. Carling, D.; Clarke, P.R.; Zammit, V.A.; Hardie, D.G. Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. Eur. J. Biochem. 1989, 186, 129–136. [PubMed] 10. Cheung, P.C.; Salt, I.P.; Davies, S.P.; Hardie, D.G.; Carling, D. Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem. J. 2000, 346 (Pt 3), 659–669. [CrossRef][PubMed] 11. Davies, S.P.; Helps, N.R.; Cohen, P.T.; Hardie, D.G. 50-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein -2C alpha and native bovine -2AC. FEBS Lett. 1995, 377, 421–425. [PubMed] 12. Oakhill, J.S.; Chen, Z.P.; Scott, J.W.; Steel, R.; Castelli, L.A.; Ling, N.; Macaulay, S.L.; Kemp, B.E. beta-Subunit is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK). Proc. Natl. Acad. Sci. USA 2010, 107, 19237–19241. [CrossRef][PubMed] 13. Zhang, Y.L.; Guo, H.; Zhang, C.S.; Lin, S.Y.; Yin, Z.; Peng, Y.; Luo, H.; Shi, Y.; Lian, G.; Zhang, C.; et al. AMP as a low-energy charge signal autonomously initiates assembly of AXIN-AMPK-LKB1 complex for AMPK activation. Cell Metab. 2013, 18, 546–555. [CrossRef][PubMed] 14. Hardie, D.G. AMPK: A target for drugs and natural products with effects on both diabetes and cancer. Diabetes 2013, 62, 2164–2172. [CrossRef][PubMed] 15. Hardie, D.G.; Ross, F.A.; Hawley, S.A. AMP-activated protein kinase: A target for drugs both ancient and modern. Chem. Biol. 2012, 19, 1222–1236. [CrossRef][PubMed] 16. Sullivan, J.E.; Carey, F.; Carling, D.; Beri, R.K. Characterisation of 50-AMP-activated protein kinase in human liver using specific peptide substrates and the effects of 50-AMP analogues on enzyme activity. Biochem. Biophys. Res. Commun. 1994, 200, 1551–1556. [CrossRef][PubMed] 17. Woods, A.; Salt, I.; Scott, J.; Hardie, D.G.; Carling, D. The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett. 1996, 397, 347–351. [CrossRef] 18. Suter, M.; Riek, U.; Tuerk, R.; Schlattner, U.; Wallimann, T.; Neumann, D. Dissecting the role of 50-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J. Biol. Chem. 2006, 281, 32207–32216. [CrossRef][PubMed] 19. Li, Y.; Cummings, R.T.; Cunningham, B.R.; Chen, Y.; Zhou, G. Homogeneous assays for adenosine 50-monophosphate-activated protein kinase. Anal. Biochem. 2003, 321, 151–156. [CrossRef] 20. Li, J.; Smith, G.P.; Walker, J.C. Kinase interaction domain of kinase-associated protein phosphatase, a phosphoprotein-binding domain. Proc. Natl. Acad. Sci. USA 1999, 96, 7821–7826. [CrossRef][PubMed] 21. Durocher, D.; Henckel, J.; Fersht, A.R.; Jackson, S.P. The FHA domain is a modular phosphopeptide recognition motif. Mol. Cell. 1999, 4, 387–394. [CrossRef] 22. Yaffe, M.B.; Cantley, L.C. . Grabbing phosphoproteins. Nature 1999, 402, 30–31. [PubMed] 23. Zhang, L.; Lee, K.C.; Bhojani, M.S.; Khan, A.P.; Shilman, A.; Holland, E.C.; Ross, B.D.; Rehemtulla, A. Molecular imaging of Akt kinase activity. Nat. Med. 2007, 13, 1114–1119. [CrossRef][PubMed] Methods and Protoc. 2018, 1, 3 10 of 10

24. Allen, M.D.; Zhang, J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem. Biophys. Res. Commun. 2006, 348, 716–721. [CrossRef][PubMed] 25. Nyati, S.; Young, G.; Ross, B.D.; Rehemtulla, A. Quantitative and Dynamic Imaging of ATM Kinase Activity. Methods Mol. Biol. 2017, 1596, 131–145. [PubMed] 26. Tsou, P.; Zheng, B.; Hsu, C.H.; Sasaki, A.T.; Cantley, L.C. A fluorescent reporter of AMPK activity and cellular energy stress. Cell Metab. 2011, 13, 476–486. [CrossRef][PubMed] 27. Neumann, D.; Woods, A.; Carling, D.; Wallimann, T.; Schlattner, U. Mammalian AMP-activated protein kinase: Functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr. Purif. 2003, 30, 230–237. [CrossRef] 28. Li, X.; Wang, L.; Zhou, X.E.; Ke, J.; de Waal, P.W.; Gu, X.; Tan, M.H.; Wang, D.; Wu, D.; Xu, H.E.; et al. Structural basis of AMPK regulation by adenine nucleotides and glycogen. Cell Res. 2015, 25, 50–66. [CrossRef][PubMed] 29. Scott, J.W.; Ling, N.; Issa, S.M.; Dite, T.A.; O’Brien, M.T.; Chen, Z.P.; Galic, S.; Langendorf, C.G.; Steinberg, G.R.; Kemp, B.E.; et al. Small molecule drug A-769662 and AMP synergistically activate naive AMPK independent of upstream kinase signaling. Chem. Biol. 2014, 21, 619–627. [CrossRef][PubMed] 30. Willows, R.; Sanders, M.J.; Xiao, B.; Patel, B.R.; Martin, S.R.; Read, J.; Wilson, J.R.; Hubbard, J.; Gamblin, S.J.; Carling, D. Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells. Biochem. J. 2017, 474, 3059–3073. [CrossRef][PubMed] 31. Rajamohan, F.; Reyes, A.R.; Frisbie, R.K.; Hoth, L.R.; Sahasrabudhe, P.; Magyar, R.; Landro, J.A.; Withka, J.M.; Caspers, N.L.; Calabrese, M.F.; et al. Probing the enzyme kinetics, allosteric modulation and activation of alpha1- and alpha2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators. Biochem. J. 2016, 473, 581–592. [CrossRef][PubMed] 32. Scott, J.W.; Galic, S.; Graham, K.L.; Foitzik, R.; Ling, N.X.; Dite, T.A.; Issa, S.M.; Langendorf, C.G.; Weng, Q.P.; Thomas, H.E.; et al. Inhibition of AMP-Activated Protein Kinase at the Allosteric Drug- Promotes Islet Release. Chem. Biol. 2015, 22, 705–711. [CrossRef][PubMed] 33. Cool, B.; Zinker, B.; Chiou, W.; Kifle, L.; Cao, N.; Perham, M.; Dickinson, R.; Adler, A.; Gagne, G.; Iyengar, R.; et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 2006, 3, 403–416. [CrossRef] [PubMed] 34. Xiao, B.; Sanders, M.J.; Carmena, D.; Bright, N.J.; Haire, L.F.; Underwood, E.; Patel, B.R.; Heath, R.B.; Walker, P.A.; Hallen, S.; et al. Structural basis of AMPK regulation by small molecule activators. Nat. Commun. 2013, 4, 3017. [CrossRef][PubMed] 35. Kurumbail, R.G.; Calabrese, M.F. Structure and Regulation of AMPK. EXS 2016, 107, 3–22. [PubMed]

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