gene productJuvenile hormone binding and ligand-dependent gene regulation Ken Miura, Masahito Oda, Sumiko Makita and Yasuo Chinzei Department of Medical Zoology, School of Medicine, Mie
Trang 1gene product
Juvenile hormone binding and ligand-dependent gene regulation Ken Miura, Masahito Oda, Sumiko Makita and Yasuo Chinzei
Department of Medical Zoology, School of Medicine, Mie University, Tsu City, Japan
Insect development and reproduction are regulated by
two classes of lipid-soluble hormones, the ecdysteroids
and juvenile hormones (JHs) The ecdysteroids activate
target genes through a heterodimeric receptor complex
composing the ecdysone receptor and ultraspiracle
(USP) proteins, both of which are members of the
nuc-lear steroid⁄ thyroid ⁄ retinoid receptor superfamily [1]
During insect development, ecdysteroids induce molting
while JH determines the nature of each molt by
modu-lating the ecdysteroid-induced gene expression cascade
[2–4] In addition, in adult insects, JH has a wide
variety of actions related to reproduction, including
oogenesis, migratory behaviour and diapause [2,5,6] The mode of molecular action of JH, however, is still obscure [7] JHs are a family of esterified sesquiterpe-noids, whose lipid-soluble nature has suggested action directly on the genome through nuclear receptors such
as ecdysteroids and the vertebrate steroid⁄ thyroid ⁄ reti-noid hormones [5,8] although actions of JH through the cell membrane are also documented [9,10]
Many attempts have been made to identify nuclear
JH receptors Jones and Sharp [11] showed that JH III binds to the Drosophila USP protein, which is a homo-logue of the vertebrate retinoid X receptor, promoting
Keywords
juvenile hormone; juvenile hormone
receptor; Methoprene-tolerant; Drosophila;
transcription factor
Correspondence
K Miura, Department of Medical Zoology,
School of Medicine, Mie University,
Edobashi 2-174, Tsu514-8507, Japan
Fax: +81 59 231 5215
Tel: +81 59 231 5013
E-mail: k-miura@doc.medic.mie-u.ac.jp
(Received 27 October 2004, revised 20
December 2004, accepted 4 January 2005)
doi:10.1111/j.1742-4658.2005.04552.x
Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction As yet the molecular modes of action of JH are poorly understood The Methoprene-tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking We have now examined the product of Met(MET) in terms of its binding to JH and ligand-dependent gene regu-lation In vitro synthesized MET directly bound to JH III with high affinity (Kd¼ 5.3 ± 1.5 nm, mean ± SD), consistent with the physiological JH concentration In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-dependent activation of a reporter gene Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness:
JH III JH II > JH I > methoprene; compounds which are only structur-ally related to JH or JHA did not induce any activation Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of
JH These results suggest that MET may function as a JH-dependent tran-scription factor
Abbreviations
Ahr, aryl hydrocarbon receptor; Arnt, Ahr nuclear translocator; bHLH, basic helix-loop-helix; DBD, DNA binding domain; DCC, dextran-coated charcoal; EGFP, enhanced green fluorescent protein; JH, juvenile hormone; JHA, synthetic analogue of JH; Met, Methoprene-tolerant gene; MET, Met protein; PAS, period-aryl hydrocarbon receptor ⁄ aryl hydrocarbon receptor nuclear translocator-single-minded; Per, Drosophila period clock protein; Sim, Drosophila single-minded protein; SFM, serum-free medium; TNT, coupled in vitro transcription⁄ translation; UAS, upstream activating sequence; USP, ultraspiracle protein.
Trang 2its homodimerization, but the concentrations of JH
required are several orders of magnitude higher than
its physiological titre [12] In transiently transfected
cultured cells, a high dose of JH led to transcriptional
activation through the binding of USP to a DNA
response element upstream of a core promoter [13] In
other insect species, gene regulation by JH through
DNA sequences resembling nuclear hormone response
elements has been reported [14–16], suggesting the
involvement of nuclear receptor family members in JH
signaling
Because the strict regulation of JH titre in the insect
body is crucial [17], the application of exogenous JH
or analogues (JHAs) can disrupt normal development,
and a number of JHAs have been synthesized and used
as insecticides as well as research tools The genetic
and biochemical studies on resistance to JHA
insecti-cides have led to the implication of another class of
transcriptional regulator in JH signaling Wilson and
coworkers examined the resistance mechanism of
Dro-sophila to the JHA, methoprene, and isolated mutant
lines of flies that are resistant to morphogenetic and
lethal effects of natural JH or methoprene [18] The
allele responsible for this resistance, named
Metho-prene-tolerant (Met), encodes a basic helix-loop-helix
(bHLH)-PAS protein, MET [19] The bHLH-PAS
fam-ily comprises transcriptional regulator proteins that are
key players in a wide array of developmental and
physiological pathways such as neurogenesis, circadian
rhythms, hypoxia response, and toxin metabolism
[20,21] PAS is an acronym from the initial members
of the family: Drosophila period clock protein (Per),
vertebrate aryl hydrocarbon receptor (Ahr, also known
as dioxin receptor)⁄ Ahr nuclear translocator (Arnt),
and Drosophila single-minded protein (Sim) [22,23]
The bHLH-PAS transcription factors share a
com-mon overall structure The bHLH domain is located
near the N terminus The basic region binds to a
con-sensus palindromic hexanucleotide E-box (CANNTG)
[24] or its derivatives [25,26] The HLH domain allows
these proteins to form a hetero- or homodimer The
bHLH domain is followed by PAS-1 and PAS-2
domains, which are used for dimerization between PAS
proteins, small molecule binding, and also for binding
to non-PAS proteins The C-terminal half residues,
which are not well conserved, harbour transcription
activation⁄ repression domains [20] These structural
features are found in MET [19] Met mutant flies
exhi-bit low JH binding affinity in fat body cytosolic
extracts while an 85-kDa protein seems to be
respon-sible for this binding [27,28] Localization of MET in
Drosophila tissues is exclusively nuclear [29] Met
females show reduced oogenesis [30], and the males
have some defects in reproduction [28,31] The Met null mutant flies are viable, showing that Met is not a vital gene, but this might be explained by redundancy provi-ded by cognate genes [30] These observations suggest the involvement of MET in at least one pathway of JH signaling The direct evidence, however, is still lacking: does JH bind directly to MET?; does MET function as
a JH-dependent transcriptional regulator?
We have now examined the binding of radiolabeled
JH III to MET protein Using a heterologous system
in Drosophila S2 cells, we have characterized ligand-dependent gene regulation by MET Our results suggest that MET may function as a JH-dependent transcription factor
Results
MET binds to JH III with high affinity The MET protein was obtained by using coupled
in vitro transcription⁄ translation (TNT) reaction The production of the full-length polypeptide was confirmed
by analysing the product of reaction in the presence of
35S-methionine by SDS⁄ PAGE and autoradiography (Fig 1) As a negative control, mock-programmed lysate was processed in parallel As evident in the figure, the principal product had the expected full-length molecular mass of 79 kDa Faster migrating minor pro-tein bands are also visible, which were not eliminated by the addition of protease inhibitor mixture in the reac-tion (data not shown) Then, the programmed lysate was used as a protein source for binding assay by the dextran-coated charcoal (DCC) method As seen in Fig 2, specific binding of3H-labeled JH III to the TNT protein showed saturable profile The experiment was repeated three times, and the Kd value by Scatchard analysis was calculated to be 5.3 ± 1.5 nm (mean ± SD) By these experiments, it was demonstrated that MET binds to JH III directly with a nanomolar Kd value although we do not rule out the possibility that factors in the rabbit reticulocyte lysate may influence binding The specific binding was competed away by 100-fold molar excess of cold JH III (data not shown)
MET regulates transcription in a JH-dependent manner
The Met gene product was examined for its transacti-vation ability The binding sequence motif of MET is presently uncertain In addition, it is unknown whether MET functions as a homo- or heterodimer So, we utilized a heterologous approach with the yeast GAL4–DBD (DNA binding domain) fusion⁄ UAS
Trang 3(upstream activating sequence) system The GAL4–
DBD possesses a zinc finger that directs
homodimeri-zation and binding to UAS elements, and a potent
nuclear localization sequence [32,33] By using this
system, we were able to assess the transactivation
potential of MET independently of its dimerization
properties or nuclear localization signals The MET
protein was expressed in S2 cells as a fusion with
GAL4–DBD, and a luciferase reporter construct
pos-sessing five tandem copies of the UAS in its regulatory
region was used In this system, the effect of JH on
transcription from the reporter gene was tested
As shown in Fig 3, when an empty expression
vec-tor was transfected, the addition of 5 lm JH III to the
culture medium caused no elevation of reporter
activ-ity over that of controls given the vehicle ethanol
slightly elevated the reporter activity, but did not show
any JH dependency Next, only the GAL4–DBD was
expressed In this case, the reporter activity was
eleva-ted about twofold above the empty vector control in
either the presence or absence of JH III, indicating
that the GAL4–DBD translocates into the nucleus and
functions as a moderate, constitutive activator of
transcription in a JH-independent manner The
GAL4–DBD–MET fusion in the presence of ethanol did not bring about any enhanced reporter activity relative to the empty vector control, but when JH III was added, the reporter activity was elevated about fivefold over the case of the empty vector control or the case of the GAL4–DBD–MET with ethanol This activation by JH III can also be described as about twofold when compared to the case of GAL4–DBD with JH III This indicates that MET has transactiva-tion domain(s), and its transactivatransactiva-tion functransactiva-tion is JH dependent It is noteworthy that in the absence of
JH III the MET moiety of the fusion protein repressed the moderate transactivation produced by GAL4– DBD This suggests that unliganded MET may func-tion as a transcripfunc-tional repressor
Fig 1 Autoradiogram of TNT lysate programmed with Met cDNA.
The TNT reaction was performed with 400 ng PCR fragment
con-taining T7 promoter and Met full ORF in the presence of 35
S-methio-nine A portion of the lysate was separated by 10% SDS ⁄ PAGE
and autoradiographed A mock-programmed lysate was run in
paral-lel Molecular mass markers are shown at the left An arrowhead
indicates the position of full-length MET (79 kDa).
Fig 2 MET binds to JH III with high affinity (A) Binding of 3 H-labe-led JH III to MET MET was obtained by TNT reaction and subjec-ted to DCC assay with 3 H-labeled JH III as described in Experimental procedures Specific binding was calculated by sub-tracting the counts of mock-programmed lysates from those of cor-responding programmed lysates The specific binding is shown in the figure (B) Scatchard analysis of JH III binding to MET The K d
value was calculated from the slope of the regression line These analyses were performed three times and representative data are shown.
Trang 4Ligand specificity of transactivation by MET
If MET represents a JH-dependent transcription
fac-tor, it should show stringent ligand specificity To test
this, several compounds that are structurally related to
JH or JHA but show no JH activity were examined in
the GAL4-MET fusion⁄ UAS system The effects of
these potential ligands on the reporter activity are
shown as fold induction by dividing the activity
obtained with the pAcGAL4–DBD–Met by that in
negative controls using empty pAc vectors (Fig 4) As
is evident here, addition of squalene, farnesol, farnesyl
acetate and geraniol at a final concentration of 5 lm
did not result in any activation of the reporter JH III,
however, again brought about enhanced reporter
activity Interestingly, a JHA) methoprene ) showed
weaker ligand activity than JH III Thus, the
trans-activation exerted by MET shows stringent ligand
specificity apparently related to JH activity, ruling out
nonspecific transactivation by lipid-soluble compounds
Dose–responses of natural JHs and JHA on MET
transactivation
The binding assay showed that MET has a nanomolar
level Kd for JH III and we used several potential
lig-ands at 5 lm in the experiments described above If
MET functions as a JH-dependent transcription factor,
it should respond to nanomolar levels of ligand,
con-sistent with its high affinity for JH III Here, we tested
three natural JHs, JH I, JH II, and JH III, and the
JHA methoprene in varying concentrations using the
GAL4-MET fusion⁄ UAS transfection assay (Fig 5) The effects on the reporter activity are shown as fold induction as in Fig 4 Every compound tested showed ligand activity on transactivation, nearing saturation at
500 nm while showing only marginal increase at 5 lm Among these, JH III, which is one of the native JHs
of Drosophila, was found to be the most effective over the range of concentrations tested Of note is that
JH III was conspicuously active in the range of 5–50 nm, whereas the other JHs or JHA showed only
Fig 4 Ligand specificity of gene activation by MET The transfec-tion assay was carried out as described in Fig 3 with pAcGAL4– DBD-Met or an empty pAc5.1 ⁄ V5-His A vector as expression con-structs S2 cells were incubated with several different compounds indicated at a concentration of 5 l M Activities are shown as fold induction by dividing the activity obtained with the fusion-expres-sing construct by that in negative controls ufusion-expres-sing empty vectors (mean ± SD) The mean value obtained in ethanol controls is taken
as unity.
Fig 3 MET regulates transcription in a JH-dependent manner S2
cells were transfected with several different expression constructs
(vector pAc5.1 ⁄ V5-His A, pAcMet, pAcGAL4–DBD, or pAcGAL4–
DBD-Met) together with reporter (pG5luc) and coreporter (pRL-tk)
constructs After incubation in either the presence or absence of
5 l M JH III for 24 h, cells were harvested and subjected to dual
luciferase assay Luciferase activities are shown normalized to that
of the coreporter (mean ± SD).
Fig 5 Dose–response curves for transactivation by MET with JHs
and JHA JH I (r), JH II (n), JH III (m), methoprene (x) were
inclu-ded in the culture media in the range from 5 n M to 5 l M after trans-fecting S2 cells with the expression (pAcGAL4–DBD-Met or empty pAc5.1 ⁄ V5-His A), reporter (pG5luc) and coreporter (pRL-tk) con-structs Fold induction was calculated as in Fig 4.
Trang 5slight effects The other native JH of Drosophila,
JH-bisepoxide [34] was not tested The induction activities
are in the following order: JH III JH II >
JH I > methoprene The most effective transcriptional
activation produced by Drosophila MET with its native
JH species further supports the putative role of MET
as a JH-dependent transcription factor We should
mention here that JHs are highly sticky to glass or
plastic surfaces [35] and would be adsorbed by pipette
tips, test tubes or culture dishes Thus, the effective
concentrations of these compounds would be lower
than the values indicated in Fig 5 These data, thus,
indicate that the threshold activity concentration of
JHs is reasonably low in this transient transfection
system
Localization of MET in S2 cells
In the transfection assays described above, MET was
fused to GAL4–DBD, which has a nuclear localization
sequence To test the subcellular localization of MET,
we used a fusion to enhanced green fluorescent protein
(EGFP), which does not have a nuclear localization
sequence S2 cells were transfected with the expression
plasmid pAcMET–EGFP together with the reporter
and coreporter constructs used above After
trans-fection, cells were incubated for 24 h in the presence
or absence of JH III, then observed by Nomarski
DIC (differential-interference contrast) or fluorescence
microscopy (Fig 6) In both cases, the fluorescence of
the fusion proteins was seen in the nucleus In these
experiments the use of cultured cells allows for
com-plete depletion of JH These observations are
consis-tent with the previous report in vivo [29] and rule out
the ligand-dependent nuclear translocation reported
for the Ahrs of vertebrates [36] Then, how is JH
transported to the nucleus? A process such as
verteb-rate retinoid transport including cellular
retinol-bind-ing protein [37] may be involved
Discussion
From its identification as a Drosophila gene responsible
for resistance to morphogenetic and toxic effects of JH
and JHAs, the Met gene product has been implicated
to have an involvement in JH reception Previous
works on Met do not contradict the hypothesis that
MET may be a component of a JH-dependent
tran-scriptional regulator complex Direct evidence,
how-ever, for the immediate interaction with JH and
involvement in gene regulation is lacking To test this
hypothesis, we chose S2 cells as experimental material
because the use of cultured cells would be
advanta-geous for examining ligand-dependent gene regulation and JH responses in this cell line have been reported [38–40]
Our principal new contributions are: (a) demonstra-tion of direct, reversible binding of JH III to MET; (b) demonstration of its JH-dependent transactivation potential The former was enabled by the use of cou-pled in vitro transcription and translation, as we had experienced difficulty in obtaining soluble preparations
of full-length bHLH-PAS proteins from mosquitoes using prokaryotic expression systems (K Miura, unpublished data) The binding of JH III by MET showed high affinity with a nanomolar Kd value, and was competed away by an excess of cold JH III
In the present study, MET was tethered to a promo-ter by using the GAL4–DBD fusion⁄ UAS reporter system In this heterologous system, the MET fusion
Fig 6 Subcellular localization of MET in S2 cells S2 cells were transfected with pAcMET–EGFP together with the reporter and coreporter constructs After transfection for 5 h, cells were incuba-ted with either 0.5 l M JH III or ethanol for 24 h Then, cells were fixed and observed by Nomarski DIC or fluorescence microscopy Horizontal bars represent 10 lm The results shown are representa-tive of two independent experiments.
Trang 6exhibited specific ligand-dependent activation of a
reporter gene placed downstream of the UAS The
MET fusion responded only to JH or the JHA
metho-prene while compounds that are structurally related
but hormonally inactive elicited no response Among
compounds tested, JH III was the most effective
lig-and, even at nanomolar concentrations, which is in
accordance with its nature as one of Drosophila’s
native JHs The typical range of concentration for
JH In insect haemolymph is 0.3–180 nm [41] Further,
the maximal JH titre in the Drosophila life cycle is
5–7 pmolÆg)1 wet weight [12], which would correspond
to 25–35 nm in the haemolymph, assuming that
haemolymph occupies one-fifth of the body weight In
view of these physiological JH titres, it is thus notable
that in this study JH III was found to be
overwhelm-ingly active in the physiological range of 5–50 nm over
the other JHs or JHA The ligand-dependent
trans-activation profile exhibited by MET clearly rules out
the possibility that it is simply a JH binder, like
cyto-solic JH binding proteins, and suggests that it might
play a role in JH signaling in vivo
Recently, Wozniak et al [42] have reported the
con-formational changes of recombinant Drosophila USP
exposed to several different farnesoid compounds
including natural JHs They have shown that JH III
and JH I at 100 lm elicit the conformational changes
to a similar degree whereas JH II is the least effective
Furthermore, by using Drosophila white puparial
bio-assay they have demonstrated that the biochemical
dif-ferences in the three JHs mentioned above parallel the
respective biological activity For example, in
preven-tion of adult emergence the 50% effective doses
(ED50s) for JH I–III are 153, 678 and 143
pmolÆpupa-rium)1, respectively Another report describes that the
ED50of methoprene is 5 pmolÆpuparium)1in the same
assay and that Met mutant flies are more resistant to
another JHA, S31183, than the parental fly stock,
sug-gesting the involvement of the Met locus in this
resist-ance [43] Based on these studies, the order of efficacy
of these compounds in this bioassay seems to be
metho-prene JH III ‡ JH I > JH II On the other hand,
the order was JH III JH II > JH I > methoprene
in our transfection assay Methoprene is the most
effective in the former and the least effective in the
latter We do not find this surprising as methoprene is
often highly active over naturally occurring JHs when
applied topically For example, the early trypsin gene
of Aedes aegypti is upregulated by JH, and low doses
of methoprene, but higher doses of its native JH III
are required to restore its expression in the ligated
abdomens [44] The higher efficacy of methoprene in
these bioassays may be due to its higher resistance to
enzymatic degradation and possible higher penetration through the cuticle than natural JHs
Another point is that JH I has been shown to be more active than JH II in the white puparial assay [42] whereas JH II is more active in our transfection assay The difference between these two studies is the concen-trations of JH used Wozniak et al [42] used supra-physiological concentrations in both biochemical and biological assays whereas we tested JHs or JHA at much lower range of concentrations In our assay JH I and JH II were similarly much less effective than
JH III in the physiological range (5–50 nm) while these differences were somewhat obscured at higher doses, although JH II was still more effective than JH I At present we do not have data to explain this discrep-ancy Possibly, there might be more than one pathway
of JH signaling underlying in the white puparial bio-assay, one mediated by USP and another by MET
In the reporter assays, we noted that unliganded MET repressed the intrinsic activation function pos-sessed by GAL4–DBD Although GAL4–DBD is believed to lack transactivation domains [33], it showed moderate transactivation potential in Dro-sophila S2 cells in this study The nuclear localization
of MET [29] was confirmed by our finding that the MET–EGFP fusion is concentrated in the nuclei of transfected S2 cells In addition, GAL4–DBD has a nuclear localization sequence Therefore, it is reason-able to consider that the GAL4–DBD fusion of MET sits on the UAS of the reporter construct even in the absence of ligand, and that the MET moiety is respon-sible for the observed repression In the case of verteb-rate Ahr, a multimeric complex including hsp90 anchors the unliganded Ahr in the cytoplasm, thereby preventing its transactivation function [36] Upon lig-and binding, Ahr translocates to the nucleus lig-and forms
a transcription factor complex with Arnt In fact, the C-terminal portion of Ahr fused to GAL4–DBD has been shown to act as a constitutive activator of gene regulation [45] Contrary to this, MET exists in the nucleus even in the absence of ligand Upon ligand binding, it becomes a transcriptional activator This resembles the ligand-dependent activation that has been shown in the activation function-2 of many nuc-lear hormone receptors [46,47], rather than the case of the vertebrate Ahr whose activation function is regula-ted by its subcellular localization
Two questions arise here as to whether MET func-tions as a homo- or heterodimer, and as to what DNA sequences are responsible for the binding of this tran-scriptional regulator complex These questions are related since DNA-binding specificities of bHLH-PAS proteins are determined by their dimerization properties
Trang 7[48] For example, the dioxin receptor complex
Ahr⁄ Arnt heterodimer binds to TNGCGTG [25] Ahr
recognizes the 5¢-half-site TNGC, while Arnt
recogni-zes the 3¢-half-site GTG Arnt is also capable of
forming a homodimer that recognizes a consensus
palindromic E-box sequence, CACGTG [48]
Dro-sophilaSim protein forms a heterodimer with Tango (a
Drosophila Arnt-like protein) and binds to ACGTG
core sequence [49] Thus, DNA binding specificities of
bHLH-PAS dimers are dependent upon the dimer
con-figuration while Arnt or Tango always recognize the
GTG motif In the present study, we used the GAL4–
DBD fusion of MET in transfection assays Under
these conditions MET is likely to behave as a
homo-dimer because of its overexpression and because of
dimerization interfaces provided by the GAL4–DBD
moiety Therefore, the natural dimerization partner
and binding sequence of MET are unknown at present
Since the bHLH domain of MET shows relatively high
similarity to vertebrate Arnts [19], the use of the
con-sensus sequence CACGTG may be a good starting
point to answer these questions
Based on the framework by Wilson and coworkers,
our results have further supported the notion that
MET may function as a JH-dependent transcription
factor In further studies, identification of its target
genes will help elucidate its in vivo function Molecular
dissection of MET and structural studies may lead to
the development of new biologically active JHA and
new strategies for pest management
Experimental procedures
JHs, JHA and related compounds
JH I and JH II were obtained from SciTech JH III was
from Sigma The JHA, methoprene was a gift from Otsuka
Chemicals Co Ltd Squalene, farnesol and geraniol were
from Sigma Farnesyl acetate was from Aldrich
cDNA cloning of Met
Total RNA was isolated from S2 cells as described
previ-ously [50] First-strand cDNA was synthesized by
Super-script reverse tranSuper-scriptase II (Invitrogen) with oligo dT
primer, and used as a template for RT–PCR The cDNA
containing a full ORF of Met was amplified by 30 cycles of
PCR using a proofreading polymerase (long and accurate
Taq polymerase, Takara) with the primer pair based on the
published sequence [19]: 5¢-GCCGAATTCCAACATGGC
AGCACCAGAGACGGG-3¢; 5¢-GCCTCTAGATCATCG
CAGCGTGCTGGTCAG-3¢ The amplified products were
purified, digested and subcloned into EcoRI and XbaI sites
of pBluescript II (Stratagene), and the identity of the cDNA clone was confirmed by sequencing
Binding assay The DCC assay was carried out as described [51] Full-length MET was prepared by a TNT T7 Quick for PCR DNA Kit (Promega) A cDNA template for the TNT reac-tion was prepared by PCR using the Met cDNA inserted downstream of the T7 promoter site of pBluescript II After PCR, the cDNA fragments containing the T7 promoter were purified by a QIAquick PCR purification kit (Qiagen) The TNT reaction was carried out as follows: 20 lL lysate was programmed by 400 ng of the cDNA fragment in a total vol-ume of 25 lL, and the reaction mixture was kept at 30C for 90 min A reaction in the presence of 35S-methionine was performed in parallel, and the lysate was analysed by SDS⁄ PAGE and autoradiography to confirm the production
of a polypeptide with the expected size The DCC assay used the TNT lysate as a protein source Each reaction mixture included 25 lL of the programmed lysate, 74 lL of buffer C (20 mm Tris⁄ HCl pH 7.9, 5 mm magnesium acetate, 1 mm EDTA, 1 mm dithiothreitol) and 1 lL of variable amounts
of 3H-labeled JH III (specific activity: 17.5 CiÆmmol)1, PerkinElmer) in ethanol in a polyethylene glycol-coated glass tube The mixture was incubated at 22C for 90 min This was followed by the addition of 5% DCC suspension, gentle mixing for 2 min and centrifugation for 1 min The supernatant was collected into a scintillation vial, decolo-rized overnight with 2 mL 30% H2O2, and counted by scin-tillation Mock-programmed lysates were incubated with the corresponding amounts of3H-labeled JH III and processed
in parallel with the programmed lysates, and the counts obtained were taken as nonspecific binding Specific binding was obtained by subtracting the counts of mock-pro-grammed lysates from those of corresponding promock-pro-grammed lysate The addition of esterase or protease inhibitors in the binding reaction mixture did not affect binding values (data not shown) Saturation curves were obtained, and Kdvalues were calculated by the method of Scatchard [52]
Plasmids The plasmid pAcGAL4–DBD-Met, which expresses a fusion protein of MET possessing the yeast GAL4–DBD toward the N terminus, was constructed as follows: a full-length Met cDNA fragment having overhangs of EcoRV and XbaI sites was prepared by PCR and subcloned into pBIND vector (Promega); the cDNA fragment containing the fused ORF of GAL4–DBD and Met was amplified by PCR and subcloned into NotI and XbaI sites of pAc5.1⁄ V5-His A vector (Invitrogen) The location of the junction was confirmed by sequencing A control plasmid pAc-GAL4–DBD was constructed by inserting the pAc-GAL4–DBD
Trang 8region of the pBIND vector into the pAc5.1⁄ V5-His A
vector Another control vector pAcMet was prepared by
subcloning the full ORF of Met into the pAc5.1⁄ V5-His A
vector An empty pAc5.1⁄ V5-His A vector was used as a
negative control The reporter plasmid pG5luc, which
con-tains five GAL4 binding sites (UAS) upstream of the firefly
luciferase gene, was from Clontech The coreporter plasmid
pRL-tk, which expresses Renilla reniformis luciferase,
was from Promega The plasmid pAcMET–EGFP, which
expresses a fusion protein of MET possessing the EGFP
polypeptide in the C terminus, was constructed by
transfer-ring the fused ORF from pEGFP-N1 vector (Clontech)
to pAc5.1⁄ V5-His A vector The in-frame nature of the
junction was confirmed by sequencing
Cell culture and transfection
Drosophila S2 cells [53,54] were cultured in Drosophila
Serum-Free Medium (SFM, Invitrogen) The cells were
see-ded at a density of 2.5· 105cells per well of 24-well plates
one day before transfection 2 lL of lipofectin (Invitrogen)
per each well was mixed with 25 lL of SFM and incubated
for 40 min at room temperature One-hundred and fifty
nanograms of DNA (50 ng each of expression, reporter and
coreporter plasmids) per well was mixed with 25 lL of
SFM, and this was combined with the lipofectin⁄ SFM
mix-ture and incubated for another 15 min Then, this was
mixed with 200 lL of SFM and overlaid onto S2 cells in
each well This was followed by 5 h incubation at 27C,
and the transfection mixture was replaced by 250 lL of
SFM either containing natural JH (JH I, JH II and JH III),
JHA methoprene, related compounds (squalene, farnesol,
geraniol and farnesyl acetate), or solvent ethanol The cells
were incubated at 27C for another 24 h, lysed and
subjec-ted to the dual luciferase assay (Promega) in a luminometer
(Turner Designs, Model TD-20⁄ 20) The reporter activity
was shown as relative luciferase activity by normalizing the
reporter activity to the coreporter activity Where indicated,
the effect of test compounds were shown as fold induction
by dividing the reporter activity obtained with the
pAc-GAL4–DBD-Met by that in negative controls using empty
pAc vectors The transfection assay was done at least three
times independently in triplicate, and the reproducibility
was confirmed The values of relative luciferase activity in
each transfection assay fluctuated a little, but tendency was
always reproducible In Results, representative data are
shown in the respective figures
For the observation of the MET–EGFP fusion proteins,
1· 106
of S2 cells were seeded on a 35-mm glass-bottomed
dish (Matsunami Glass, #D110400) 1 day before
transfec-tion The pAcMET–EGFP was transfected together with
the reporter and coreporter constructs as described above
so as to mimic the transfection conditions of the reporter
assays After 24 h in either the presence or absence of
0.5 lm JH III, the cells were washed three times in NaCl⁄ Pi
on the dishes, fixed in 4% (w⁄ v) paraformaldehyde in NaCl⁄ Pi for 30 min at room temperature, rinsed twice in NaCl⁄ Pi, and observed directly by an inverted microscope (Nikon, Model TE300) equipped with Nomarski DIC and fluorescence optics
Acknowledgements
We thank Drs Takahiro Shiotsuki and Tetsuro Shi-noda for the help on DCC assay; Dr Gerard R Wyatt for reading the manuscript This work was supported
in part by a grant-in-aid for Scientific Research (C)
to KM (15580049) from the Ministry of Education, Science, Culture, and Sports of Japan
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