PRIORITY PAPER Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1 Jean-Pierre Bouly 1 , Baldissera Giovani 1,2 , Armin Djamei 3 , Markus Mueller 3 , Anke Zeugner 1 , Elizabeth A. Dudkin 4 , Alfred Batschauer 3 and Margaret Ahmad 1,4 1 Universite ´ Paris VI, Paris, France; 2 Service de Bioe ´ ne ´ rge ´ tique, Commissariat a ` l’Energie Atomique Saclay, Gif-sur-Yvette, France; 3 Plant Physiology, Phillips-Universitaet Marburg, Germany; 4 Penn State University, Media, PA, USA Cryptochromes are blue-light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV-damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence simi- larity in both catalytic and cofactor-binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity asso- ciated with Arabidopsis cry1 protein purified from a baculo- virus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of crypto- chrome depleted in flavin and/or misfolded. Autophospho- rylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes. Keywords: cryptochrome; photolyase; blue light; photo- receptor; autophosphorylation. Cryptochromes are blue-light photoreceptors found in plants and animals implicated in multiple blue-light depend- ent signaling pathways [1]. These include de-etiolation responses such as inhibition of hypocotyl elongation and anthocyanin accumulation in plants, leaf and cotyledon expansion, transition to flowering, or regulation of blue- light regulated genes. In animal systems, cryptochromes have been shown to play a role in circadian rhythms, either directly as components of the circadian pacemaker in mouse [2,3] or, in Drosophila, more indirectly by feeding light information into the circadian clock [4]. The defining characteristics of cryptochromes are N-terminal domains with marked similarity to photolyases [4–6], a class of flavoprotein that catalyse repair of UV-damaged DNA via light-dependent electron transfer reactions [7]. Crypto- chromes bind similar cofactors to photolyases, yet lack DNA repair activity [8–10], suggesting evolution of novel activities to explain their role in signaling. Interestingly, although sharing many sequence similarities and apparent functional analogy, plant and animal cryptochromes appear to have evolved independently from different ancestral photolyases, with animal cryptochromes sharing greater sequence similarity to type 6-4 photolyases and plant cryptochromes more similar to type I microbial photolyases [5]. A further defining characteristic of both plant and animal cryptochromes are C-terminal extensions, not found in photolyases, which are essential for a number of cryptochrome functions. Ectopic expression of the C-terminal domain of plant cry1, for example, results in a constitutive de-etiolation response in the absence of light, leading to the suggestion that cryptochromes may function via a light-dependent conformational change that renders these photoreceptors accessible to proteins implicated in cellular signaling pathways [11]. The identification of several such signaling molecules, in particular cop1, which binds to the C-terminal of cry1 both in vivo and in vitro, lends support to such a notion [12,13]. Animal crypto- chromesalsohavebeenshowntointeractdirectlywith cellular signaling intermediates, notably components of the circadian clock [14,15]. Photolyases function by light-dependent electron transfer subsequent to excitation of the flavin cofactor, either involving the pyrimidine dimer of UV-damaged DNA or through a separate pathway involving intraprotein electron transfer [16]. It has recently been shown that a similar light- dependent intramolecular transfer reaction, involving both tyrosine and tryptophan radicals, also occurs in crypto- chrome [17]. However, the means whereby such an intramolecular electron transfer reaction, lasting only milliseconds, can play a role in signaling and result in interaction of the photoreceptor with putative downstream signaling intermediates that are not permanently bound to the photoreceptor remains a puzzle. In this work we present a novel ATP-binding and autophosphorylation activity, not found in photolyases, but present in both plant and animal cryptochromes, and discuss this activity in light of a possible role in signaling of the cryptochrome photoreceptors. Correspondence to M. Ahmad, Universite ´ Paris VI, UMR-CNRS 7632, Tour 53 E 5, Casier 156, 4, Place Jussieu, 75252 Paris Cedex 05, France. Fax: + 33 144272916, Tel.: + 33 144272916, E-mail: ahmad@ccr.jussieu.fr (Received 4 February 2003, revised 8 April 2003, accepted 4 June 2003) Eur. J. Biochem. 270, 2921–2928 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03691.x Experimental procedures Purification of Arabidopsis and human cry1 from insect cells Full-length Arabidopsis cry1 retaining an N-terminal His 6 affinity tag was expressed and purified to apparent homogeneity from Sf21 insect cells on nickel columns as described [8]. The purified protein is a yellow protein that binds flavin in the oxidized form. When Co 2+ affinity resin (Clontech Laboratories, Palo Alto, CA) was used instead of Ni 2+ , lysates turned bright yellow during the binding reaction due to release of flavin from the cry1 protein to the surrounding medium. The purified bound protein, in contrast, proved colourless and the absorption spectrum was obtained in a Beckmann DU7400 spectrophotometer. Human cry1 protein con- taining an N-terminal His 6 affinity tag was expressed and purified over Ni 2+ affinity columns by the same procedure. ATP-agarose affinity chromatography Assays involving purified cryptochrome from baculovirus were performed as follows: after two washes with 1 mL of buffer A (25 m M Hepes, pH 7.4; 150 m M NaCl; 1 m M dithiothreitol and 60 m M MgCl 2 ), 0.1 mL of adenosine 5¢-triphosphate immobilized on cross linked 4% beaded agarose (Sigma, cat. no. A2767) were incubated with 4 lgof cryptochrome in buffer A for 2 h at 4 °C. After incubation, the beads together with bound protein were washed three times with 1 mL of buffer B (buffer A containing 500 m M NaCl). The bound cryptochrome was eluted by incubation with 100 lL of buffer C (buffer A containing 20 m M ATP) for 1 h at 4 °C. The eluted supernatant was analyzed by SDS/PAGE. For plant cryptochrome binding assays, total protein extracts were prepared from a dark-grown Arabidopsis cell culture: 1 mL (2 mg) of total protein extract in extraction buffer (50 m M Tris/HCl, pH 7.5; 10 m M NaCl; 5 m M MgCl 2 ; 2.5% glycerol; plant protease inhibitor mix, Sigma) was incubated with 0.2 mL of ATP-agarose (Sigma, cat. no. A2767) for 1 h at 4 °C. After incubation, the beads were washed four times with 1 mL of wash solution (50 m M Tris/HCl, pH 7.5; 100 m M NaCl; 5 m M MgCl 2 ) and the protein eluted by incubation in 120 lL elution buffer (wash buffer containing 20 m M ATP) for 15 min at 4 °C. Proteins from the different fractions were subjected to SDS/PAGE and immunoblot analysis with antibodies against cry1 and against histone as a negative control. Direct photo-crosslinking of nucleotides to cry1 Cry1 protein samples were mixed with 40 lCi [a- 32 P]ATP diluted to a final concentration of 2 l M in unlabelled ATP; 50 m M Hepes, pH 7.0; 20 m M MgCl 2 and the reaction mixture (50 lL)wasincubatedoniceinthedarkfor 20 min. Twenty-five microlitres was removed and exposed to short-wavelength UV light for 10 min on ice. After treatment, the samples were subjected to SDS/PAGE and autoradiography. Quantification of ATP binding in terms of affinity and stoichiometry Cry1 protein (3 lg) was bound to the Ni 2+ or Co 2+ resin and subjected to stringent washes as for the purification procedure. Instead of eluting the bound protein, samples were subsequently incubated for 2 h at 20 °Cin50m M Hepes (pH 7.0), 20 m M MgCl 2 and protease inhibitors with increasing concentrations of ATP containing [a- 32 P]ATP at constant specific activity. After five washes with buffer containing 50 m M Tris/HCl (pH 7.5), 500 m M NaCl and 10 m M imidazole, the amount of ATP bound either to the immobilized cry1 or remaining free in solution was deter- mined by liquid scintillation counting and K d 1 have been determined. Phosphorylation reactions and phosphoamino acid analysis Phosphorylation reactions were carried out at 25 °Cin buffer containing 4 lg of cryptochrome, 5 lCi of [c- 32 P]ATP or [a- 32 P]ATP diluted to a final concentration of 200 l M in unlabelled ATP, 50 m M Hepes, pH 7.0, 20 m M MgCl 2 or 20 m M MnCl 2 or 20 m M CaCl 2 for 1 h. The reactions were stopped by addition of SDS/PAGE sample buffer and labelled proteins were visualized on SDS gels by Coomassie staining followed by autoradiography. For phosphoamino acid analysis, 32 P-labelled crypto- chrome was treated as described by Hardin & Wolniak [18]. Light-dependent phosphorylation reactions Phosphorylation reactions were carried out at 25 °Cin buffer containing 2 lg of cryptochrome; 50 m M Hepes, pH 7.0; 5 lCi of [c- 32 P]ATP diluted to a final concentration of 2 l M in unlabelled ATP and 2 l M MgCl 2 .Samples were maintained in dark for 10 min, 10 min in white light, or alternatively illuminated for 5 min in the absence of substrate (ATP and MgCl 2 ) followed by 10 min further illumination in the presence of ATP and MgCl 2 (5 + 10min). The reactions were performed in the presence of 10 m M 2-mercaptoethanol, 1 m M KI or 0,003% of H 2 O 2 . Reactions were stopped by addition of SDS/PAGE sample buffer and labelled proteins were visualized on SDS gels by Coomassie staining. After cutting out the cry1 band, c- 32 P incorporation was determined by liquid scintillation count- ing; c- 32 P incorporated in the sample kept in the dark (with 2-mercaptoethanol) is taken as the reference. All crypto- chrome preparations for light-induction studies were freshly purifiedandusedafter24hdarkadaptationat4°C without prior freezing of the sample. Results To identify possible novel biochemical activities associated with cryptochrome, Arabidopsis cry1 (Atcry1) was expressed in a baculovirus system and purified to near homogeneity by Ni 2+ affinity column chromatography as previously des- cribed [6].In such preparations flavin is bound in the oxidized form and the loosely associated methenyltetrahydrofolate (MTHF) 2 secondary cofactor is apparently lost in the purification process [19]. We have determined that if Atcry1 2922 J P. Bouly et al. (Eur. J. Biochem. 270) Ó FEBS 2003 protein expressed in Sf21 cell extracts is purified on Co 2+ instead of Ni 2+ affinity columns, both the flavin cofactor and MTHF are lost 3 , and the cryptochrome apoprotein depleted in both cofactors can be isolated (Fig. 1A,B). We have used these preparations to investigate possible cryptochrome- associated phosphorylation activity in vitro and observed strong labelling of a band corresponding to Atcry1 in the presence of MgCl 2 and c- 32 P ATP. This activity is found only in cryptochrome preparations retaining flavin chromo- phore (native protein), but not in flavin-depleted protein preparations under the identical assay conditions (Fig. 1C). Cryptochromes have been identified as substrates for protein kinases in several systems [20,21] and have no homology to known protein kinases themselves. Therefore, to eliminate the formal possibility that trace quantities of contaminating kinases may copurify with native Atcry1 and thereby cause the observed labeling reaction, we have directly investigated the ATP binding activity of crypto- chrome by several complementary approaches. In the first approach, purified Atcry1 protein was found to bind quantitatively and completely to ATP agarose affinity columns, no cry1 proteins were lost in the different columns washes and all the protein bound to the column could be completely and specifically eluted with ATP, providing evidence that Atcry1 has ATP binding site(s) (Fig. 2A). In another approach, UV cross linking studies were performed in the presence of a- 32 P labelled ATP (Fig. 2B), which can bind to cryptochrome but not radioactively phosphorylate it. Such photo cross-linking studies are a classic method for the identification of ATP binding sites in proteins, due to the tighter association of ATP to the protein upon photo- activation of the purine [22]. After incubation with Atcry1 protein, the majority of [a- 32 P]ATP is not retained after electrophoresis on denaturing SDS gels although, after sufficiently long exposure times, a faint band of residual bound ATP can be visualized on autoradiographs at the position of the Atcry1 protein. After UV-treatment, the degree of labeling is significantly elevated, consistent with tighter binding and crosslinking of the nucleotide directly to the Atcry1 protein (Fig. 2B). Next, the relative affinity of cryptochrome for ATP was examined by quantitative methods under nondena- turing conditions, and the dissociation constant (K d )was determined for ATP binding (Fig. 2C). Atcry1 protein from insect cell extracts was immobilized on Ni 2+ or Co 2+ affinity columns and subjected to stringent washing as in the purification procedure. However, instead of eluting the protein, immobilized samples were exposed to [a- 32 P]ATP at constant specific activity and varying concentrations of ATP, and the proportion of bound ATP ascertained by scintillation counting. The binding curve obtained shows that a higher amount of ATP is bound by native Atcry1 compared with the flavin- depleted form of Atcry1 at all concentrations of ATP tested. Scatchard plot analysis is consistent with a single ATP binding site per cryptochrome. The proportion of ATP binding is calculated as 0.4 molecules ATP bound per molecule of native Atcry1, with a binding affinity of dissociation constant K d of 19.8 l M . These data are not consistent with a minor contaminant protein being responsible for ATP binding or with nonspecific binding, both of these values being well within the range of such data obtained with known ATP binding protein with high and specific affinity for ATP [23,24]. Flavin-depleted Atcry1 showed somewhat reduced binding affinity and stoichiometry (K d of 25.1 l M and a calculated 0.19 ATP molecules per molecule cry1 protein; values derived from data in Fig. 2C). Possibly the observed reduction in binding affinity may be due to a degree of cry1 protein misfolding in such flavin-depleted preparations, as crystal structure analysis reveals flavin is in contact with multiple amino acids throughout both Escherichia coli photolyase and a recently characterized novel cryptochrome protein [25], and may thereby help to stabilize the tertiary structure. Finally, to confirm that our findings are not limited to our recombinant cryptochrome preparations, native Atcry1 protein was assayed from crude extracts of Arabidopsis cell cultures and subjected to ATP agarose affinity column chromatography. It was found that all the native cry1 (and Fig. 1. Purification and phosphorylation of Arabidopsis cry1 from insect cells. (A) Twenty micrograms of Atcry1 protein purified to apparent homogeneity by either Ni 2+ or Co 2+ affinity column chromatography was resolved on SDS gels and stained with Coomassie Blue. (B) Absorption spectra of Atcry1 protein (0.7 mgÆmL )1 ) purified from either Ni 2+ or Co 2+ affinity columns. Bound flavin is in the oxidized form. (C) Phos- phorylation of native or flavin-depleted cry1 protein. Samples were labeled with [c- 32 P]ATP as indicated in Experimental procedures and run on SDS polyacrylamide gels. The left panel represents the Coomassie stained protein samples, in the right panel the autoradiogram of these samples is shown. Ó FEBS 2003 Cryptochrome autophosphorylation (Eur. J. Biochem. 270) 2923 also cry2, data not shown) protein bound quantitatively to ATP agarose affinity columns, and could be completely and specifically eluted with ATP (Fig. 2D). From these data it can be concluded that crypochrome quantitatively and specifically binds ATP, which is a necessary condition if it is to undergo an autophosphorylation reaction. To further characterize the phosphorylation reaction, it was determined that labelling of Atcry1 in the presence of [c- 32 P]ATP vastly exceeds that of [a- 32 P]ATP (Fig. 3A), indicating that there is transfer of the labeled c-phosphate of ATP to the Atcry1 protein and thereby a phosphorylation reaction as opposed to simply nucleotide binding. Labeling also occurs in the presence of [c- 32 P]GTP (not shown). The phosphorylation reaction requires magnesium and does not occur in the presence of either MnCl 2 or CaCl 2 , neither is it stimulated by CaCl 2 in the presence of MgCl 2 .The phosphotransfer reaction has a requirement that flavin is bound to the molecule (Fig. 3A); in a prior study of Atcry1 phosphorylation, the flavin proved not to be bound and for this reason autophosphorylation was not detected [21] (M. Ahmad, unpublished data). Because flavin stabilizes the conformation of the closely related E. coli photolyase in addition to participating in catalysis [26], possibly the conformational and/or catalytic requirements for ATP binding are less stringent than those for the phosphotransfer reaction. Interestingly, none of several possible substrates for classic protein kinases were phosphorylated by crypto- chromes, including histones, casein, and MBP (MAP kinase substrate) (not shown). Therefore cryptochrome may only be capable of phosphorylating itself and not other substrates. To identify the phosphorylated amino acid(s) of crypto- chrome, native cryptochrome was labeled and separated on SDS/PAGE gels, which were subsequently immersed in acid, base, or neutral solutions prior to autoradiography. The radioactivity in the cry1 4 band was retained under acid but not base conditions (not shown), indicating that the phosphate link is base labile (characteristic of phospho- serine and phospho-threonine but not phospho-tyrosine). To provide definitive evidence of the labeled residue, phosphorylated cryptochrome was submitted to phospho- amino acid analysis [18]. As shown in Fig. 3B, only labeled phospho-serine was detected, and the nonradiolabeled standards (phospho-serine, -threonine, and -tyrosine) were clearly visible as well defined spots by ninhydrin detection. Therefore, cryptochrome undergoes autophosporylation Fig. 2. ATP-binding activity of purified cryptochromes. (A) ATP agarose affinity purification of Arabidopsis cryptochrome visualized on Coomassie- stained gels. Lane 1, purified protein before binding reaction; lane 2, supernatant after incubation with the ATP binding resin (unbound Atcry1); lane 3, supernatant after three washes with 500 m M NaCL;lane4,elutedsamplewith20m M ATP; lane 5, remaining cryptochrome on resin after elution (as determined by boiling of the resin in SDS/PAGE sample buffer subsequent to the last elution). (B) Direct photocross linking of nucleotides. Atcry1 protein samples were electrophoresed before (–) and after (+) UV crosslinking treatment, visualized by Coomassie staining and subsequent autoradiography of dried gels. (C) Scatchard plot analysis for the binding of ATP to cry1 of data from the insert; insert binding of ATP to cry1 immobilized on Ni 2+ or Co 2+ affinity columns. (D) ATP binding activity of plant cry1. Total protein extracts from an Arabidopsis cell culture were incubated with ATP-agarose and bound protein eluted with 20 m M ATP. Protein fractions were subjected to SDS/PAGE and immunoblot analysis using antibodies specific for cry1 and for histone as a control. The following amounts of protein were loaded per lane: lane 1 (total protein extract), 200 lg; lane 2 (supernatant), 200 lg; lane 3 (eluate), 40 lg. After elution, no cry1 signal could be observed in the ATP- agarose fraction. 2924 J P. Bouly et al. (Eur. J. Biochem. 270) Ó FEBS 2003 exclusively at serine residue(s). In order to test the degree of phosphorylation, time course experiments have been per- formed. We found that the autophosphorylation reaction was saturable after about 30 min under in vitro conditions (Fig. 3C). It has been reported that the Atcry2 protein is rapidly phosphorylated in vivo in response to blue-light irradiation [27], and also that cryptochrome responses appear to be under redox control in Arabidopsis cell culture systems [28]. To relate our in vitro phosphorylation data to these possible early signaling events of the photoreceptor in vivo,we investigated Atcry1 autophosphorylation in response to light and redox state. A short irradiation of purified cry1 protein previously maintained in darkness for 24 h significantly stimulated the autophosphorylation reaction (Fig. 4A). Interestingly, irradiation by light for 5 min prior to addition of substrate resulted in substantially increased phosphory- lation as compared to simultaneous irradiation (Fig. 4A, 5+10min light), suggesting that preillumination results in a long-lived activation of the receptor consistent with changes in redox state of the flavin. Flavin antagonists such as KI that have been found to inhibit redox reactions mediated by other flavoproteins [29] or oxidizing agents such as H 2 O 2 abolished light stimulation of the phosphorylation reaction (Fig. 4B,C), indicating that cryptochrome autophosphory- lation is regulated in vitro by both redox state and light. It has been determined that, as for cry2 [24], a rapid blue- light dependent shift in gel mobility due to phosphorylation of cry1 protein occurs in Arabidopsis seedlings (A. Batscha- uer, unpublished observations). However, we have deter- mined that both phosphorylated and unphosphorylated baculovirus-expressed cry1 protein migrate at the same mobility as dark-adapted cry1 from etiolated Arabidopsis seedlings (data not shown). Therefore, the shift in mobility resulting from blue-light dependent phosphorylation of cry1 (and presumably also cry2) in vivo is a result of labeling by external plant protein kinases, likely at different sites in the protein than those involved in the autophosphorylation reaction. Cryptochromes from plant and animal systems differ in apparent evolutionary origins, animal cryptochrome being most similar to 6-4 photolyases whereas plant crypto- chromes apparently evolved from the class I CPD photo- lyases [5]. Nevertheless, there is considerable similarity between these two classes of signaling molecule. To establish whether animal cryptochrome may likewise contain ATP binding and autophosphorylation activity, human cry1 protein (Hscry1) was expressed in insect cell culture with an N-terminal His 6 tag as for Arabidopsis cry1. Purified Hscry1 protein was isolated by nickel affinity column chromato- graphy and the identity of the expressed protein confirmed by Western blot analysis to antibody specific for the C-terminal domain of mouse and human cry1 protein (Fig. 5). Like Arabidopsis cry1, Hscry1 protein was shown to bind to an ATP agarose affinity column and could be eluted specifically with ATP. Furthermore, Hscry1 protein is also phosphorylated in the presence of MgCl 2 and [c- 32 P]ATP. Thus, ATP binding and autophosphorylation activity is apparently retained in both plant and animal cryptochromes. Discussion We have characterized an ATP binding and autophospho- rylation reaction that is intrinsic to the Arabidopsis cry1 Fig. 3. Phosphorylation activity associated with Atcry1. (A) Phos- phorylation reactions were performed as described in Experimental procedures under the indicated buffer and/or purification conditions. (B) Phosphoamino acid analysis. Phosphoamino acid standard spots visualized with ninhydrin are circled. The origin is the lowest spot. (C) Time course of Atcry1 7 autophophorylation. The mean of three experiments of time course is shown. At equilibrium, the stoichiometry of Atcry1 autophopshorylation was between 0.1 and 0.15 mol phos- phate per mol Atcry1. Ó FEBS 2003 Cryptochrome autophosphorylation (Eur. J. Biochem. 270) 2925 blue-light photoreceptor. This phosphorylation activity is unexpected as cryptochrome shares no amino acid sequence similarity with known protein kinases or ATP binding proteins, although precedents exist for other signaling molecules with unpredicted ATP binding [23] and autophosphorylation activity [23,30]. Furthermore, cryptochromes do not phosphorylate classic substrates such as histones, casein, or MBP, and thereby do not appear to act as protein kinases. Photolyases, the apparent evolutionary ancestors of cryptochromes, have not been reported to bind ATP; nor have we detected auto- phosphorylation activity in purified E. coli photolyase preparations (M. Ahmad, unpublished results). Because photolyases contain nucleotide binding pockets located in close association with the catalytic flavin cofactor [31], it seems plausible that a novel ATP-binding and auto- phosphorylation activity, regulated by light and redox state of the cofactor, could have evolved from such an enzyme. Nevertheless, as there is very little difference between the amino acid sequence of cryptochromes and the most closely related photolyases it is quite a puzzle how such a profound transformation in activity could result. We have determined that the N-terminal domain of Atcry1 with homology to photolyases by itself is sufficient to undergo the autophosphorylation reaction in vitro (M. Ahmad, unpublished data). Therefore, the transformation in enzymatic activity from photolyases to cryptochromes has occurred with just a few amino acid substitutions. Further experiments to explore this trans- formation should be extremely interesting from a struc- tural, mechanistic, and evolutionary point of view for both function of photolyases and cryptochromes. How can this autophosphorylation reaction be reconciled with the novel signaling function of cryptochromes? It has been proposed that cryptochromes may function by under- going intramolecular conformational changes that make them accessible to degradative enzymes [32,33] or expose C-terminal effector domains to substrate [11–13]. It has furthermore been shown that Atcry1 undergoes intra- molecular light-dependent electron transfer reactions in vitro [17], and that reduction/oxidation reactions involving electron transfer through the conserved tryptophan path- way are critical for light dependent activities of CRY in organisms such as Drosophila,orXenopus [34–36]. How- ever, intramolecular redox reactions alone are too rapid to result in stable conformational changes of the photorecep- tors, as they occur within fractions of a second and are unlikely to involve profound changes in structures of cofactors. Unless cryptochrome were permanently associ- ated with its substrate (as is the case with photolyases), it is difficult to reconcile intramolecular electron transfer reac- tions with stable changes in photoreceptor conformation Fig. 4. Effect of light and redox agents on cry1 autophosphorylation. Samples were maintained in dark for 10 min, 10 min in white light, or alternatively illuminated for 5 min in the absence of substrate (ATP and MgCl 2 ) following by 10 min further illumination in the presence of ATP and MgCl 2 (5 + 10min). The reactions were performed (A) in the presence of 10 m M 2-mercaptoethanol. (B) In the presence 1 m M potassium iodide (KI). (C) In the presence 0.003% of H 2 O 2 . Error bars represent the SE of three independent experiments. Autoradiogram shows auto- phosphorylation activity of one experiment. Fig. 5. ATP-binding and autophosphorylation activity associated with hCRY1. A cDNA clone comprising the entire coding region of human CRY1 gene with the addition of an N-terminal His 6 tag-encoding linker was cloned and expressed in baculovirus expression system as described for Atcry1 [8] 8 . Subsequent to purification on Ni resin, a band of approximately 67 kDa was eluted which crossreacted by Western blot analysis with anti-mCRY1 Ig diluted 500· (made to a 21 amino acid peptide sequence within the C-terminus of CRY1) from Alpha Diagnostic International, San Antonio, TX, ref. CRY11-A. Purified Hscry1 was incubated together with ATP agarose resin and allowed to bind as described for AtCry. Bound and unbound fractions were visualized on polyacrylamide gels followed by Western blot analysis with anti-mCry1 Ig. Lane Pre, sample prior to addition of ATP agarose beads; Post, remaining Hscry1 protein in sample subsequent to incubation with ATP-agarose; Eluted, sample eluted specifically from the column, after the indicated washes (see Experimental procedures), with 20 m M ATP. An autoradiogram shows autophosphorylation activity associated with purified human cry1 under identical assay conditions to AtCry1. 2926 J P. Bouly et al. (Eur. J. Biochem. 270) Ó FEBS 2003 that would be long-lived enough to enable activated photoreceptor to find and interact with potential substrate molecules. In the present work we show that the autophosphoryla- tion of Atcry1 is sensitive both to light and the redox state of the enzyme, and that it requires flavin. Hence intramole- cular phosphorylation, regulated by light-induced changes in redox state, may be a mechanism to effect long-lived, covalent intramolecular conformational changes important for the signaling function of cryptochromes. The observa- tion that this activity has been identified in both plant and animal cryptochromes, which apparently evolved from separate ancestors, lends support to the functional signifi- cance of this phenomenon. Further experiments to explore the signaling role of this autophosphorylation activity are currently in progress. Acknowledgements We are indebted to Dr Paul Galland for valuable advice, Nabil Lounis for help with phosphorylation studies, Alain Picaud for the phospho- amino acid analysis, Andre ´ Klarsfeld for the gift of HsCRY1 antibody, and to members of the plant science laboratory (LPDP) at the University of Paris for helpful discussions. This work was funded by a fellowship from the C.E.A to B. G., grants from the CNRS (Atipe Blanche and SdV) to M. A., and from the Deutsche Forschungsgeme- inschaft (Ba985/7-2) to A. B. References 1. Ahmad, M. (2003) Cryptochromes and flavoprotein blue-light photoreceptors. In Handbook of Photobiology and Biochemistry (Nalwa, H.S., ed.). Academic Press, San Diego, in press. 5 2. Van der Horst, G.T.J., Muitjtjens, M., Kobayashi, K., Takano, R.,Kanno,S.,Takao,M.,deWit,J.,Verkerk,A.,Eker,A.P.M., van Leenen, D., Buijis, R., Bootsma, D., Hoeijmakers, J.H.J. & Yasui, A. (1999) Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. 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