Proc. NatI. Acad. Sci. USAVol. 86, pp. 3469-3473, May 1989Biochemistry

Affinity labeling of Avena phytochrome with ATP analogs(plant photoreceptor/nucleotide binding site/polycation-stimulated protein kinase/photobiology/photoaffinity labeling)

YUM-SHING WONG AND J. CLARK LAGARIAS*Department of Biochemistry and Biophysics, University of California, Davis, CA 95616

Communicated by Eric E. Conn, February 13, 1989 (received for review September 2, 1988)

ABSTRACT The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phy-tochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh,D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochromephotoreceptor possesses enzymatic activity. A prerequisite forprotein kinase function is the presence of an ATP binding site.Here we present evidence for a nucleoside triphosphate bindingsite(s) in the phytochrome molecule. Two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5'-triphosphate, were used to affinity label purified Avena phy-tochrome. Labeling with both reagents is stimulated by thepolycations poly(Lys75,Ala25) and histone H1. Coincubationwith ATP inhibits the polycation-stimulated labeling of phy-tochrome. In similar experiments GTP, CTP, UTP, ADP, andpyrophosphate, but not adenosine or AMP, also preventphotoaffinity labeling of phytochrome.

The chromoprotein phytochrome plays a central role in plantphotoperception throughout the entire life cycle (1, 2). Owingto its unique ability to adopt two photointerconvertibleforms, the red-absorbing Pr form and the far-red-absorbingPfr form, phytochrome mediates adaptation to changes in thelight environment at the cellular and organismal level. At thebiochemical level, little is known of the signal-transductionpathway that is initiated by the formation of Pfr, the generallyaccepted physiologically active species of phytochrome.Since structural differences between the Pr and Pfr forms ofphytochrome may indicate a functionally significant interac-tion with an as yet unidentified receptor molecule, thepurified photoreceptor has been the object of extensivebiochemical analyses (3). During the course of one suchstudy, we reported that purified Avena phytochrome prepa-rations contain polycation-stimulated protein kinase activity(4). These studies raised the possibility that this proteinkinase activity represents a functional property of the phy-tochrome molecule (4, 5). This hypothesis is further sup-ported by the observations that phytochrome binds to Ciba-cron blue-agarose (6) and that the primary structure ofphytochrome and the catalytic domains of known proteinkinases have regions of sequence similarity (5, 7).The ATP analogs 5'-p-fluorosulfonylbenzoyladenosine

(FSBA) (8, 9) and 8-azidoadenosine 5'-triphosphate (N3ATP)(10, 11) have been successfully used to probe nucleosidetriphosphate binding sites of a wide variety of proteins,including protein kinases, ATPases, cytoskeletal proteins,and enzymes involved in polynucleotide synthesis and repair.In the present report, we show that the affinity reagent[14C]FSBA and the photoaffinity reagent [a-32P]N3ATP ef-fectively label purified Avena phytochrome. Moreover, con-sistent with the hypothesis that phytochrome is a polycation-stimulated protein kinase, we show that labeling of phy-tochrome with both reagents is enhanced in the presence of

polycations. The specificity of affinity labeling was demon-strated by competition experiments with nucleoside triphos-phates. These results provide evidence for the presence of anucleoside triphosphate binding site(s) within the phyto-chrome photoreceptor.

MATERIALS AND METHODSAffinity Labeling of Phytochrome with [14C]FSBA. Phy-

tochrome was purified from 5-day-old etiolated oat seedlings(Avena sativa L. cv. Garry) according to published proce-dures (12). Before affinity labeling, phytochrome prepara-tions were exhaustively dialyzed at 50C into a 50 mMEpps/Tris buffer (pH 7.8) containing 1 mM EDTA and 25%(vol/vol) ethylene glycol. These preparations exhibited spe-cific absorption ratios (i.e., the ratio of Aw to A280 for Pr)between 0.90 and 1.0. To initiate affinity labeling, a 5.0 mMstock solution of [14C]FSBA (48.9 mCi/mmol; New EnglandNuclear; 1 mCi = 37 MBq) in dimethyl sulfoxide was addedto the phytochrome solutions (0.5-1.0 mg/ml) to give finalconcentrations of 0.5 mM and 0.2-0.5 mg/ml for FSBA andphytochrome, respectively. FSBA concentrations were de-termined using a molar absorption coefficient at 232 nm of1.88 x 104 M-1.cm-1 (13). When indicated, poly(Lys75,Ala25)[poly(Lys-HBr,Ala) 3:1, Sigma catalog no. P1151] was addedas a 0.2 mg/ml stock solution in 20 mM Tris HCl (pH 7.8) toa final concentration of 40 jig/ml. When poly(Lys75,Ala25)was not present, the final volume was adjusted with 20 mMTris HCI (pH 7.8). For competition experiments whereMgATP was also added, its concentration was estimatedspectrophotometrically by using a molar absorption coeffi-cient at 259 nm of 1.5 x 104 M-1.cm-1 (14). The final mixtureswere incubated at 30°C for 30 min unless otherwise indicated.Reactions were terminated by addition of an equal volume ofNaDodSO4/PAGE sample buffer [125 mM Tris-HC1, pH6.8/15% (vol/vol) glycerol/5.5% (wt/vol) NaDodSO4/5%(vol/vol) 2-mercaptoethanol/0.01% bromophenol blue] fol-lowed by heating for 1 min at 100°C prior to electrophoresis.

Photoaffinity Labeling of Phytochrome with [a-32P]N3ATP.Except for photolyses, all procedures were performed underdim green light (12). A 1 mM stock solution of [a-32P]N3ATPtriethylammonium salt (18.1 Ci/mmol; from ICN) in 20 mMTris HCI, pH 7.8/1 mM MgCl2 was prepared to afford a finalspecific activity of 300-500 cpm/pmol. N3ATP concentra-tions were determined spectrophotometrically by using amolar absorption coefficient at 281 nm of 1.33 X 104M-1'cm-1 (15). Unirradiated or preirradiated control stocksolutions of Mg[a-32P]N3ATP were added to the phy-tochrome solutions to give final concentrations of 200 ,uMand 0.28 mg/ml for N3ATP and phytochrome, respectively.Preirradiation conditions were empirically determined withour light source to ensure that the MgN3ATP photolabel had

Abbreviations: FSBA, 5'-p-fluorosulfonylbenzoyladenosine; N3-ATP, 8-azidoadenosine 5'-triphosphate; Pr, red-absorbing form ofphytochrome; Pfr, far-red-absorbing form of phytochrome.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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3470 Biochemistry: Wong and Lagarias

been fully photolyzed. In some experiments, poly(Lys75,Ala25) or histone H1 (Sigma type V-S) was also added to thereaction mixture to give a final concentration of 40 Ag/mlprior to photolysis. For competition experiments, magne-sium salts of all nucleotides and pyrophosphate were em-ployed. Concentrations of all nucleotides were determinedspectrophotometrically (14). Photolyses were performed at00C in open Eppendorf microcentrifuge tubes. The lightsource (a General Electric mercury spot lamp equipped witha Corning CVX.DRL filter) was held at a distance of 15 cmfrom the sample, a condition that gave a fluence rate atsurface of the sample of 173 uW/cm2 as determined with acalibrated thermopile (16). After photolysis, the sampleswere diluted with an equal volume of NaDodSO4 samplebuffer, heated for 1 min at 100'C, and analyzed by NaDod-SO4/PAGE and autoradiography.NaDodSO4/PAGE, Autoradiography, and Fluorography.

NaDodSO4/PAGE was conducted in 0.8-mm slab gels com-posed of 3% stacking and 7.5% resolving gels with theLaemmli buffer system (17). Gels with 32P-labeled phy-tochrome were stained with 0.05% (wt/vol) Coomassie blueR-250 in methanol/acetic acid/water (5:1:5, vol/vol) for 1 hrand destained in methanol/acetic acid/water (5:1:5, vol/vol).Destained gels were equilibrated in 10% acetic acid contain-ing 1% (vol/vol) glycerol for 1 hr and dried onto Whatman3MM paper under vacuum at 80°C. Autoradiography wasperformed at -80°C with Kodak X-Omat AR film and aDuPont Cronex intensifying screen. Gels with 14C-labeledphytochrome were stained briefly (5 min) with Coomassieblue R-250. Destained gels were then treated with EN3-HANCE autoradiography enhancer (New England Nuclear)according to the manufacturer's instruction for fluorography.

Radiolabel Quantitation. For 32P quantitation, labeled phy-tochrome bands were excised from the dried gel and treatedwith LSC scintillation fluid (National Diagnostics, Manville,NJ), and radioactivity was measured with a Beckman LS-3133P scintillation counter (4). For 14C quantitation, labeledphytochrome bands were excised and dissolved in 0.5 ml of30% H202/concentrated NH40H (95:5, vol/vol) overnight at37°C. LSC fluid (10 ml) was added to each sample andradioactivity was quantitated by scintillation spectrometry.

Protein Assays. Phytochrome concentrations were deter-mined spectrophotometrically by using the molar absorptioncoefficient of Pr at 668 nm of 1.32 x 105 M-1.cm-1 per124-kDa subunit (18). For estimation of protein concentra-tions, the bicinchoninic acid method was used with bovineserum albumin as the protein standard (19).

RESULTSAffinity Labeling of Phytochrome with [14C]FSBA. Affinity

labeling of phytochrome with the hydrophobic reagent[14C]FSBA required addition of organic solvents to the finalmixture. For this reason, experiments were undertaken toaddress the effects of two organic solvents, ethanol anddimethyl sulfoxide, on the solubility and spectral propertiesof phytochrome. Of the two solvents, dimethyl sulfoxideproved superior, with levels of up to 10% by volume havingno measurable effect on phytochrome photoreversibility.Experiments performed with [14C]FSBA in 10% dimethylsulfoxide showed that both Pr and Pfr forms of phytochromewere labeled with this reagent (Fig. 1). Addition of poly-(Lys75,Ala25) to the reaction mixture resulted in a 2-foldstimulation of radiolabel incorporation into phytochrome(Fig. 1). The time dependence of affinity labeling of phy-tochrome with [14C]FSBA in the presence of poly(Lys75,Ala25) is illustrated in Fig. 2. ATP competition experimentsrevealed that the rate of affinity labeling ofphytochrome with[14C]FSBA was measurably reduced by coincubation withATP (Fig. 2). A separate competition experiment showed that

Prd

2

Pfr

3 4

FIG. 1. Poly(Lys75,Ala25) stimulation of affinity labeling of phy-tochrome with ['4C]FSBA. Pr (lanes 1 and 2) and Pfr (lanes 3 and 4)forms of phytochrome were incubated for 30 min at 30°C with[14C]FSBA in the absence (-) or presence (+) of poly(Lys75,Ala25).Shown here is a fluorograph of a NaDodSO4/7.5% polyacrylamidegel. The percent molar incorporation of 14C label for lanes 1-4 was2.1%, 5.4%, 2.2%, and 4.7%, respectively.

ATP and pyrophosphate at 3 mM concentrations wereequally effective in inhibiting [14C]FSBA labeling of phy-tochrome (data not shown).

Photoaffnity Labeling of Phytochrome with [a-32P]N3ATP.Except for the time-course experiment, all photoaffinitylabeling experiments with N3ATP used a 5-min UV lightpulse. This exposure was chosen to effect significant pho-tolysis of N3ATP while also minimizing irreversible damageto phytochrome. A 15-20% reduction of photoreversibilitywas determined for phytochrome preparations as Pr or Pfrafter a 5-min irradiation with UV light. This UV light doseyielded the same photoequilibrium ratio of 66% Pfr and 34%Pr irrespective of whether phytochrome was initially presentas Pr or Pfr (data not shown). While both Pr and Pfr forms ofphytochrome were labeled to similar extents with [a-32P]N3ATP under these experimental conditions, photoaffin-ity labeling was markedly stimulated by polycations (Fig. 3).The presence of either poly(Lys75,Ala25) or histone H1resulted in as much as a 50-fold increase in photoaffinitylabeling of either Pr and Pfr (Fig. 3 and Table 1). Controlexperiments, either where the mixtures were not illuminatedwith UV light or where preirradiated [a-32P]N3ATP was used,yielded little significant labeling of phytochrome (Table 1 anddata not shown).A time course of photoaffinity labeling of phytochrome in

the presence of polylysine is shown in Fig. 4. Since the aboveexperiments showed no difference in the labeling ofPr or Pfr,the time course of photoaffinity labeling of phytochrome was

24

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0

-5-

a)0~

20

16

12

a

4

00 30 60 90 120 150

Time (min)FIG. 2. Time course of polycation-stimulated affinity labeling of

phytochrome with [14C]FSBA. Phytochrome (Pr form) was incu-bated with [14C]FSBA and poly(Lys75,Ala25) in the absence (.) or

presence (A) of 5 mM ATP. Data shown represent the average of twoexperiments.

Proc. Natl. Acad. Sci. USA 86 (1989)

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Proc. Natl. Acad. Sci. USA 86 (1989) 3471

Pr Pfr 12

1 2 3 4

FIG. 3. Poly(Lys75,Ala25) stimulation of photoaffinity labeling ofphytochrome with [a-32P]N3ATP. Pr (lanes 1 and 2) and Pfr (lanes 3and 4) solutions containing [a-32P]N3ATP in the absence (lanes 1 and3, respectively) or presence (lanes 2 and 4, respectively) ofpoly(Lys75,Ala25) were irradiated with UV light. Shown here is anautoradiograph of a NaDodSO4/7.5% polyacrylamide gel.

examined only with Pr preparations. Polycation-stimulatedphotoaffinity labeling of phytochrome with [a-32P]N3ATPapproached a limiting value of 10% molar incorporation, andphotoaffinity labeling of phytochrome was effectively inhib-ited by coincubation with ATP (Fig. 4). This protectionafforded by ATP was concentration-dependent, with 5 mMATP providing almost complete protection (Fig. 5). Thenucleoside triphosphates GTP, CTP, and UTP also inhibitedpolycation-stimulated photoaffinity labeling of phytochrometo varying degrees (Table 2). ADP proved less effective thanthe nucleoside triphosphates, whereas AMP and adenosinewere ineffective as inhibitor of photoaffinity labeling ofphytochrome (Table 2). Pyrophosphate was found to be apotent inhibitor of poly(Lys75,Ala25)-stimulated photoaffinitylabeling of phytochrome with [a-32P]N3ATP.

DISCUSSIONIn a previous study, we reported that purified phytochromepreparations contain polycation-stimulated ATP-dependentprotein kinase activity (4). This observation raised the in-triguing possibility that phytochrome is a protein kinase. Ifso, it must necessarily bind ATP at a catalytic site(s). In thisstudy, we have shown that covalent modification of phy-tochrome with two different ATP analogs, FSBA and N3-ATP, is strongly stimulated by the presence of polycations.Since the structure, charge, and reactive-group chemistry ofFSBA and N3ATP are quite different, these results stronglysuggest that polycations interact with phytochrome in amanner that exposes a nucleoside triphosphate bindingsite(s). The alternative possibilities that both reagents wouldbecome activated through direct interaction with polycationsor that polycation-phytochrome interaction enhances non-specific reactions with two very different ATP analogs areless likely.ATP protection studies showed that the polycation-phy-

tochrome interaction does not merely perturb the structure of

Table 1. Polycation-stimulated photoaffinity labeling ofphytochrome with [a-32P]N3ATP

% molarCondition(s) incorporation

Experiment 1Pr ([a-32P]N3ATP

preirradiated) 0.27Pr 0.35Pr + poly(Lys75,Ala25) 6.84Pr + histone H1 6.70

Experiment 2Pr 0.10Pr + poly(Lys75,Ala25) 5.82Pfr 0.21Pfr + poly(Lys75 Ala25) 7.94

All mixtures were irradiated for 5 min with UV light. Percent molarincorporations were determined following NaDodSO4/PAGE andscintillation spectrometry of excised phytochrome bands.

00

L.0

C-L.

0C

L.a)0~

10

a

B

4

2

00 20 40 60

Time (min)FIG. 4. Time course of polycation-stimulated photoaffinity la-

beling of phytochrome with [a-32P]N3ATP. A solution containingphytochrome (Pr form), [a-32P]N3ATP, and poly(Lys75,Ala25) wasirradiated with UV light in the absence (e) or presence (A) of 5 mMATP.

phytochrome and increase the extent of nonspecific modifi-cation ofphytochrome. Coincubation with ATP was effectivein inhibiting the affinity labeling of phytochrome with bothreagents. While N3ATP photoaffinity labeling of phy-tochrome was nearly abolished by ATP coincubation, themaximum inhibition of FSBA labeling of phytochrome byATP was 50%, which indicated the lower specificity of thisreagent. The concentration dependence of the ATP compe-tition for N3ATP labeling indicated that the apparent disso-ciation constant for ATP binding to phytochrome lies be-

6

0

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-4-i

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4

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00 1 2 3 4 5 6

[ATP] (mM)FIG. 5. Effect ofATP concentration on the polycation-stimulated

photoaffinity labeling of phytochrome with [a-32P]N3ATP. A solu-tion containing phytochrome (Pr form), [a-32P]N3ATP, and poly-(Lys75,Ala25) was irradiated with UV light in the presence of variousconcentrations of ATP. The open square represents the radiolabelincorporation into phytochrome that was incubated with preirradi-ated [a-32P]N3ATP.

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3472 Biochemistry: Wong and Lagarias

Table 2. Inhibition of poly(Lys75,Ala25)-stimulated photoaffinitylabeling of phytochrome with various competitors

% molar % of % inhi-Competitor incorporation control bition

None (control) 4.76 100Adenosine 4.50 94 6AMP 4.64 97 3ADP 3.19 67 33ATP 0.91 19 81GTP 0.28 6 94CTP 0.44 9 91UTP 1.39 29 71Pyrophosphate 0.37 8 92

All mixtures were irradiated for 5 min with UV light. Competitionexperiments used 3 mM final concentrations of competitor. Exceptfor adenosine, competitors were added as Mg complexes. Percentmolar incorporations were determined as indicated in Table 1.

tween 1 and 2 mM. This value is not, however, a truedissociation constant for ATP, since the measured inhibitionof photoaffinity labeling has a complex dependency onfactors other than the dissociation constant. Reliable esti-mates of ATP binding constants by competition for N3ATPphotoaffinity labeling of phytochrome will require measure-ment of rates of inactivation; these rates are especiallydifficult to determine with photoaffinity reagents. Neverthe-less, the value of 1-2 mM lies within the concentration rangeforATP found in most cells and, therefore, provides evidenceto support a physiologically relevant interaction betweenphytochrome and ATP.Owing to the greater specificity of N3ATP, the properties

of this reagent were investigated in greater detail. Our resultsthat photoaffinity labeling with N3ATP was significantlyprevented by nucleoside triphosphates and to a lesser degreeby ADP, but not by AMP or adenosine, clearly show thatsuch protection was not caused by UV light attenuation.These data also indicate that the phosphate anhydride moietyrather than the purine moiety appears to be the more im-portant structural feature governing nucleotide-phytochromeinteraction. The strong inhibitory effect of pyrophosphate onthe N3ATP photoaffinity labeling of phytochrome supportsthis hypothesis. The inhibition by pyrophosphate is particu-larly significant in view oftwo other observations. First, 3 mMpyrophosphate also inhibited ['4C]FSBA labeling of phy-tochrome, suggesting that pyrophosphate competes with thenonionic. benzenesulfonyl fluoride moiety ofFSBA for bindingto the same region of the phytochrome polypeptide. Second,pyrophosphate is a potent inhibitor of the polycation-stimulated ATP-dependent protein kinase activity of purifiedphytochrome preparations (Y.-S.W., R. W. McMichael, Jr.,and J.C.L., unpublished data).The above discussion has emphasized arguments in sup-

port of the conclusion that phytochrome contains an ATPbinding site that is exposed by incubation with polycations.This hypothesis is strongly supported by the ATP competi-tion experiments. Alternatively, ATP coincubation couldinhibit affinity labeling of phytochrome by preventing poly-cation-phytochrome interaction rather than by competing fora nucleoside triphosphate binding site. Since all of theeffective inhibitors of phytochrome affinity labeling containnegatively charged pyrophosphate groups, an ionic associa-tion between pyrophosphate moieties and the polycation islikely. It is possible that this interaction could prevent theobserved stimulatory effects of polycations on affinity label-ing of phytochrome. In view of the low concentrations of thecompetitors that were effective in preventing phytochromeaffinity labeling, the interaction between these competitorsand the two different polycations used in these studies wouldhave to be relatively specific. For this reason, we favor the

interpretation that the polycation-stimulated labeling of phy-tochrome reflects exposure of an ATP/nucleotide bindingsite on the photoreceptor.The physiological significance of the observed affinity

labeling of phytochrome with ATP analogs remains animportant unanswered question. These results are neverthe-less consistent with our earlier proposal that the phy-tochrome photoreceptor may be a polycation-stimulatedprotein kinase (4, 5). Although phytochrome lacks the triadof glycine residues that is a characteristic structural featureof the nucleotide binding site of the mammalian proteinkinase family (20), two polypeptide sequences, Glu-Leu-Glu-Lys-Gln-Leu-Arg-Glu-Lys-Asn-Ile-Leu-Lys (resi-dues 403-415) and Asp-Leu-Lys-Leu-Asp-Gly-Leu-Ala (res-idues 606-613), found on the type 3 Avena phytochromesubunit (7) are similar to peptide sequences found within thenucleotide binding sites of known protein kinases (5, 20).These structural similarities between the catalytic domains ofknown protein kinases and sites near the central hinge regionbetween chromophore and nonchromophore domains ofphytochrome suggest that an ATP binding site may be locatedwithin this region. Preliminary limited-proteolysis experi-ments with [14C]FSBA- and [a-32P]N3ATP-labeled phyto-chrome have been performed and support this hypothesis(unpublished results). The major labeled tryptic peptidefragments obtained from samples digested as Pr were 114, 59,and 55 kDa in size, whereas labeled 84-, 69-, and 55-kDafragments were observed for samples digested as Pfr. In goodagreement with previously reported results (12), these cor-respond to major polypeptide fragments in trypsin digests ofPr and Pfr. While the precise location and number ofmodifications remain to be determined, this labeling patternindicates that (i) both chromophore and nonchromophoredomains are modified by these ATP analogs, (ii) the protease-sensitive N-terminal 10-kDa region ofAvena phytochrome isnot a major site of affinity labeling, and (iii) phytochrome isthe protein being modified by these reagents, since it isunlikely that a contaminating protein of similar molecularmass would give rise to light-dependent proteolytic digestionpatterns.

In conclusion, the presence of a polycation-dependentnucleoside triphosphate binding site of phytochrome is con-sistent with the hypothesis that phytochrome is the polyca-tion-stimulated protein kinase reported earlier. Nucleosidetriphosphate binding is, however, insufficient evidence toconclude that phytochrome has protein kinase activity.Further work is needed to resolve the issue of whether thephenomenon of polycation-stimulated nucleoside triphos-phate binding to phytochrome reflects the presence of acatalytically active ATP-phytochrome association.

We thank Robert W. McMichael, Jr., for helpful discussion and forrepeating some of the experiments. This work was supported in partby National Science Foundation Grant DMB 87-04266.

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