Báo cáo khoa học: Novel ATP-binding and autophosphorylation activity associated withArabidopsisand human cryptochrome-1 pptx

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:
(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. Nature 398, 627–630.
3. Stanewsky, R. (2002) Clock mechanisms in Drosophila. Cell Tissue
Res. 309, 11–26.
4. Ahmad, M. & Cashmore, A.R. (1993) Hy4 gene of A. thaliana
encodes a protein with characteristics of a blue-light photo-
receptor. Nature 366, 162–166.
5. Kanai, S., Kikuno, R., Toh, H., Ryo, H. & Todo, T. (1997)
Molecular evolution of the photolyase-blue-light photoreceptor
family. J. Mol. Evol. 45, 535–548.
6. Deisenhofer, J. (2000) DNA photolyases and cryptochromes. Mut
Res. 460, 143–149.
7. Carell, T., Burgdorf, L.T., Kundu, L.M. & Cichon, M. (2001) The
mechanism of action of DNA photolyases. Curr. Opin. Chem.
Biol. 5, 491–498.
8. Lin, C., Robertson, D.E., Ahmad, M., Raibekas, A.A., Jorns,
M.S., Dutton, C.L. & Cashmore, A.R. (1995) Association of fla-
vin adenine dinucleotide with the Arabidopsis blue-light receptor
CRY1. Science 269, 968–970.
9. Malhotra, K., Sang-Tae, K., Batschauer, A., Dawut, L. & Sancar,
A. (1995) Putative blue-light photoreceptors from Arabidopsis
thaliana and Sinapis alba with a high degree of sequence homology
to DNA photolyase contain the two photolyase cofactors but lack
DNA repair activity. Biochem. 34, 6892–6899.
10. Hsu, D.S., Zhao, X.D., Zhao, S.Y., Kazantsev, A., Wang, R.P.,

Todo, T., Wei, Y.F. & Sancar, A. (1996) Putative human blue-
light photoreceptors hCRY1 and hCRY2 are flavoproteins.
Biochem. 35, 13871–13877.
11. Yang, H.Q., Wu, Y.J., Tang, R.H., Liu, D., Liu, Y. & Cashmore,
A.R. (2001) The C termini of Arabidopsis cryptochromes mediate
a constitutive light response. Cell 103, 815–827.
12. Wang, H., Ma, L., Li, J., Zhao, H. & Deng, X. (2001) Direct
interaction of Arabidopsis cryptochromes with COP1 in light
control of development. Science 294, 154–158.
13. Yang, H.Q., Tang, R.H. & Cashmore, A.R. (2001) The signaling
mechanism of Arabidopsis CRY1 involves direct interaction with
COP1. Plant Cell 12, 2573–2587.
14. Rosato, E., Codd, V., Mazzotta, G., Piccin, A., Zordan, M.,
Costa, R. & Kyriacou, C.P. (2000) Light–dependent interaction
between Drosophila CRY and the clock protein PER mediated by
the carboxy terminus of CRY. Curr. Biol. 11, 909–917.
15. Shearman, L.P., Sriram, S., Weaver, D.R., Maywood, E.S.,
Chaves,I.,Zheng,B.,Kume,K.,Lee,C.C.,vanderHorst,
G.T., Hastings, M.H. & Reppert, S.M. (2000) Interacting
molecular loops in the mammalian circadian clock. Science 288,
1013–1019.
16. Aubert, C., Vos, M.H., Eker, A.P.M. & Brettel, K. (2000)
Intraprotein radical transfer during photoactivation of DNA
photolyases. Nature 405, 586–590.
17. Giovani, B., Byrdin, M., Ahmad, M. & Brettel, K. (2003) Light-
induced electron transfer in a cryptochrome blue-light photo-
receptor. Nat. Struct. Biol. 10, 489–490.
18. Hardin, S.C. & Wolniak, S.M. (1998) Low-voltage separation of
phosphoamino acids by silica gel thin-layer electrophoresis in a
DNA electrophoresis cell. Biotechniques 24, 344–346.

19. Ahmad, M., Grancher, N., Heil, M., Giovani, B., Black, R.C.,
Galland, P. & Lardemer, D. (2002) Action spectrum for crypto-
chrome-dependent hypocotyl growth inhibition in Arabidopsis
thaliana. Plant Physiol. 129, 774–785.
20. Eide, E.J., Vielhaber, E.L., Hinz, W.A. & Virshup, D.M. (2002)
The circadian regulatory proteins BMAL1 and cryptochromes
are substrates of casein kinase I epsilon. J. Biol. Chem. 277,
17248–17254.
21. Ahmad, M., Jarillo, J.A., Smirnova, O. & Cashmore, A.R. (1998)
The CRY1 blue light photoreceptor of Arabidopsis interacts with
phytochrome A in vitro. Mol. Cell. 1, 939–948.
22. Bayley, H. & Knowles, J.R. (1977) Photoaffinity labeling. Methods
Enzymol. 46, 69–114.
23. Stephenson, K. & Hoch, J.A. (2001) PAS-A domain of phos-
phorelay sensor kinase A: a catalytic ATP-binding domain
involved in the initiation of development in Bacillus subtilis. Proc.
NatlAcad.Sci.USA98, 251–256.
24. Guthapfel, R., Gueguen, P. & Quemeneur, E. (1996) ATP binding
and hydrolysis by the multifunctional protein disulfide isomerase.
J. Biol. Chem. 271, 2663–2666.
25. Brudler,R.,Hitomi,K.,Daiyasu,H.,Toh,H.,Kucho,K.,Ishi-
ura,M.,Kanehisa,M.,Roberts,V.A.,Todo,T.,Tainer,J.A.&
Getzoff, E.D. (2003) Identification of a new cryptochrome class.
Structure, function, and evolution. Mol. Cell. 11, 59–67.
26. Park, H W., Kim, S T., Sancar, A. & Deisenhofer, J. (1995)
Crystal structure of DNA photolyase from Escherichia coli.
Science 268, 1866–1872.
27. Shalitin, D. et al. (2002) Regulation of Arabidopsis cryptochrome 2
by blue-light-dependent phosphorylation. Nature 417, 763–767.
28. Long, J.C. & Jenkins, G.I. (1998) Involvement of plasma mem-

brane redox activity and calcium homeostasis in the UV-B and
UV-A/blue light induction of gene expression in Arabidopsis. Plant
Cell 10, 2077–2086.
29. Short, T.W., Porst, M. & Briggs, W.R. (1992) A photoreceptor
system regulating in vivo and in vitro phosphorylation of a pea
plasma membrane protein. Photochem. Photobiol. 55, 773–781.
30. Nishiwaki, T., Iwasaki, H., Ishiura, M. & Kondo, T. (2000)
Nucleotide binding and autophosphorylation of the clock protein
Ó FEBS 2003 Cryptochrome autophosphorylation (Eur. J. Biochem. 270) 2927
KaiC as a circadian timing process of cyanobacteria. Proc. Natl
Acad. Sci. USA 97, 495–499.
31. Komori,H.,Masui,R.,Kuramitsu,S.,Yokoyama,S.,Shibata,
T.,Inoue,Y.&Miki,K.(2001)Crystalstructureofthermostable
DNA photolyase: Pyrimidine-dimer recognition mechanism.
Proc.NatlAcad.Sci.USA98, 13560–13565.
32. Ahmad, M., Jarillo, J.A. & Cashmore, A.R. (1998) Chimeric
Proteins between cry1 and cry2 Arabidopsis blue light photo-
receptors indicate overlapping functions and varying protein sta-
bility. Plant Cell 10, 197–207.
33. Lin, C., Yang, H., Guo, H., Mockler, T., Chen, J. & Cashmore,
A.R. (1998) Enhancement of blue-light sensitivity of Arabidopsis
seedlings by a blue-light receptor cryptochrome 2. Proc. Natl
Acad. Sci. USA 95, 2686–2690.
34. Zhu, H. & Green, C.B. (2001) A putative flavin electron transport
pathway is differentially utilized in Xenopus CRY1 and CRY2.
Curr. Biol. 11, 1945–1949.
35. Ishikawa, T., Hirayama, J., Kobayashi, Y. & Todo, T. (2002)
Zebrafish CRY represses transcription mediated by CLOCK-
BMAL heterodimer without inhibiting its binding to DNA. Genes
Cells 7, 1073–1086.

36. Froy, O., Chang, D.C. & Reppert, S.M. (2002) Redox potential:
differential roles in dCRY1 and mCRY1 functions. Curr. Biol. 12,
147–152.
2928 J P. Bouly et al. (Eur. J. Biochem. 270) Ó FEBS 2003