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INTRODUCTION |
The honeybee Apis mellifera L. is a social insect, and
colony members perform various exquisite communications to maintain colony activities. Worker bees inform the other foragers of the direction and distance of a food source using dance language (1, 2),
which might require complex processing of sensory information in their
brains. Little is known, however, regarding the molecular basis of
their highly advanced behavior.
Mushroom bodies (MBs)1 are
believed to be involved in sensory integration, learning, and memory in
insects (3, 4). The honeybee MBs are well developed when compared with
those of other insects. In the honeybee, the ratio of volume of MBs to
that of whole brain is ~12%, whereas that of Drosophila
is ~2% (5). Moreover, each MB of the honeybee has two calyces
composed of two morphologically distinct types of interneurons, the
large- and small-type Kenyon cells (5-7). On the other hand, in
Drosophila, there is only one calyx, and the Kenyon cells
are morphologically indistinct (8). These observations suggest that MB
function is closely associated with the advanced honeybee behaviors.
To identify molecules involved in the highly advanced behaviors of the
honeybees, we previously used the differential display method to
identify a gene, termed Mblk-1, that is expressed
preferentially in the large-type Kenyon cells of the honeybee brain
(9). Mblk-1 encodes a novel protein consisting of 1598 amino
acid residues with significant similarity to a nuclear factor encoded
by the Drosophila melanogaster CG18389/E93 gene.
The CG18389/E93 gene was identified previously as an
ecdysone-inducible gene in the prepupal salivary gland (10) and was
reported to encode a nuclear protein that is required for
ecdysone-triggered programmed cell death during metamorphosis (11). The
expression of CG18389/E93 in the adult and the biochemical
characteristics of the protein, however, have not been examined.
Two putative DNA-binding motifs, termed RHF (region
conserved between honeybee and fruit
fly) 1 and RHF2, a nuclear localization signal, and Gln run were
conserved between Mblk-1 and Drosophila E93 protein (9).
RHF2 has significant sequence homology with proteins encoded by genes
from nematoda (a polypeptide predicted by an open reading frame of the
Caenorhabditis elegans cosmid T01C1), human (three
polypeptides predicted by open reading frames of the chromosome 4 clone
RP11-173B23 map 4, chromosome 11 clone RP11-162M10 map 11, and
chromosome 10 clone RP11-175019, respectively), mouse (12), and sea
urchin (a polypeptide predicted by an open reading frame of the
Strongylocentrotus purpuratus EST253 coelomocyte cDNA
5'-end), suggesting that the intracellular functions of these proteins
are conserved among the animal kingdom. The binding site selection
method was used to identify the preferred binding sequence of Mblk-1 as
5'-CCCTATCGATCGATCTCTACCT-3', termed MBE
(Mblk-1-binding element). Truncated
Mblk-1 protein that contains either RHF1 or RHF2 can also bind MBE but
with much lower affinity than intact Mblk-1. An in vitro
pull-down assay indicated that RHF1 and RHF2 afford homodimeric
bindings, suggesting that Mblk-1 functions as a dimer (13). These
results suggest that Mblk-1 is a transcription factor that functions in
the MB neural circuits in the honeybee brain. The molecular function of
Mblk-1, however, has not been characterized previously.
In general, long term memory formation requires protein synthesis. This
has been confirmed in animals ranging from insects and mollusks to
mammals (14). In the honeybee, the formation of long term memory
lasting 4 days requires both de novo transcription and
translation (15). Mitogen-activated protein kinase (MAPK) has a role in
long term memory in a number of different learning paradigms in
invertebrates and vertebrates (16-23). Therefore, the MAPK signaling
pathway is a good candidate involved in long term changes in neuronal
gene expression triggered by extracellular stimuli.
In the present study, we used a luciferase assay to determine whether
Mblk-1 transactivates promoters containing MBEs and can be modulated by
the Ras/MAPK pathway. The results indicated that Mblk-1 is a
transcription factor that might function in MB neural circuits directly
modulated by the Ras/MAPK pathway.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
SL-2 cells (Schneider's Line 2 cells derived
from D. melanogaster embryos) (24) were maintained in
Schneider's Drosophila medium (Invitrogen) with the
addition of heat-inactivated fetal bovine serum (Sigma), 5 mg/ml
polypeptone, and antibiotics (100 units/ml penicillin G and 0.1 mg/ml
streptomycin) (Invitrogen). The cells were grown in monolayers at
27 °C
Plasmid Construction and Mutagenesis--
A luciferase reporter
vector containing either the Hsp70Bb core promoter from
pUAST (pGL3H) or the P-element core prompter and hsp70
leader from WTP-1 (pGL3PH) was prepared by amplifying the
corresponding sequences by PCR and ligating each of these PCR
products to the pGL3-basic vector (Promega Co., Madison, WI) containing
the firefly luciferase gene. Two or six tandem copies of either MBE or
UASG (Gal4 upstream activating sequence) were subcloned
into the upstream regions (at positions
11 to
28) of pGL3H or pGL3PH.
Full-length Mblk-1 cDNA was subcloned into the multicloning
site (MCS) (BamHI-SpeI) of the actin
5C expression vector (pPac-PL) (25) and termed Mblk-1/pPac-PL. To
create a series of N-terminal deletion mutants of Mblk-1 (
1-51,
1-99, and
1-142), DNA fragments corresponding to positions from
+411, +569, and +698 to +933 of the Mblk-1 cDNA were amplified by
PCR, and each of the resulting PCR products was used to replace the
BamHI-BstPI fragment of Mblk-1/pPac-PL (
1-51,
1-99, and
1-142/pPac-PL, respectively). To create two other
N-terminal deletion mutants (
1-220 and
1-353), Mblk-1 cDNA
was digested with NcoI and BstPI or
NcoI and SgrAI and blunt-ended. These two
fragments were ligated and subcloned into the MCS
(BamHI-SpeI) of pPac-PL.
To create six series of deletion mutants, truncated Mblk-1 mutants,
cDNA fragments corresponding to +275 to +1486 (
411-1597), +1424
to +2698, +2600 to +3895, and +3890 to +5065 (
1-1204), respectively, were amplified by PCR. A cDNA fragment corresponding to +1424 to +2698 was used to replace the
NotI-SpeI fragment of
411-1597, and the
resulting insert was subcloned into pPac-PL (
809-1597/pPac-PL). A
cDNA fragment corresponding to +2600 to +3895 was used to replace
the BlpI-SpeI fragment of
809-1597, and the
resulting insert was subcloned into pPac-PL (
1208-1597/pPac-PL). A
cDNA fragment corresponding to +2600 to +3895 was used to replace the BamHI-NcoI fragment of
1-1204, and the
resulting insert was subcloned into pPac-PL (
1-775/pPac-PL). A
cDNA fragment corresponding to+1424 to +2698 was used to replace
the BamHI-BlpI fragment of
1-775, and the
resulting insert was subcloned into pPac-PL (
1-383/pPac-PL).
To create the RHF1 deletion mutant (
586-636) of Mblk-1, we utilized
a HindIII site, which was located just at the end of RHF1.
The DNA fragment that corresponds to +1691 to +2030 with an extra
nucleotide for the HindIII site at the 3'-end was amplified by PCR, and the resulting PCR product was used to replace the Asp718-HindIII fragment of the Mblk-1-(384-808)
cDNA. The insert of this plasmid was then used to replace the
NotI-BlpI fragment of the full-length Mblk-1
cDNA, and the resulting insert was subcloned into pPac-PL
(
586-636/pPac-PL). To create the RHF2 deletion mutant (
1031-1088) of Mblk-1, DNA fragments that correspond to +3188 to
+3364 and +3539 to +5071 with extra nucleotides for the
Asp718 site at the 5'- and 3'-end, respectively, were
amplified by PCR. The resulting PCR products were then digested with
Asp718, ligated to each other, subcloned, and
digested with BsgI and SpeI. The resulting
fragment was used to replace the BsgI-SpeI of the
Mblk-1-(776-1207) plasmid. The resulting plasmid was again used to
replace the BlpI-BstZ17I of the
full-length Mblk-1 plasmid and subcloned into pPac-PL
(
1031-1088/pPac-PL).
To create the Mblk-1S444A mutant, PCR was performed using the first
sense primer, 5'-CACCTCTCGCACCGCAGAGCGACAGTAGCA-3', where the underline indicates nucleotides corresponding to mutated Ala-444; the second sense primer,
5'-aaaTGATCAACCACCTCTCGCACCGCAGAG-3'; the antisense primer,
5'-CTAGGTACCGGTGAGAGCC-3'), which correspond to +1588 to +1714 of
Mblk-1; and the full-length Mblk-1 cDNA as a template. The PCR
product was subcloned, digested with BclI-Asp718, and used to replace the BclI-Asp718 of the
Mblk-1-(384-808) plasmid. This mutated Mblk-1-(384-808) plasmid was
used to replace the NotI-BlpI of the full-length
Mblk-1 plasmid and subcloned into pPac-PL (Mblk-1S444A/pPac-PL). The
pPac expression plasmids containing the actin 5C promoter
and either Ras1V12 (constitutively active Ras1) or
MAPKSem (constitutively active MAPK) cDNA were kind
gifts from Dr. T. Hsu (Medical University of South Carolina,
Charleston, SC) (26)
Transfections and Reporter Assay--
Transfection experiments
were performed essentially as described previously (27). SL-2 cells
(2-5 × 105 cells/ml) were cultured in 1 ml of
Schneider's Drosophila medium containing heat-inactivated
fetal bovine serum and 5 mg/ml polypeptone in a 12-well plate for
24 h at 27 °C to allow them to adhere to the dish, and the
medium was discarded. A mixture of plasmid DNA (0.5 µg) was incubated
with 2 µl of Cellfectin reagent (Invitrogen) in 0.1 ml of
Drosophila serum-free medium (Invitrogen) for 30 min, and
then 0.4 ml of Drosophila serum-free medium was added to
increase the volume. The resulting total mixture was added to the
adhered cells and incubated for 4 h to accomplish transfection. The medium was then replaced with fresh Schneider's
Drosophila medium containing heat-inactivated fetal bovine
serum and 5 mg/ml polypeptone, and incubation was continued. The
reporter gene activities were assayed 42-44 h later. Cells were
collected and lysed in the reporter lysis buffer (Promega Co.),
and luciferase activity in the lysate was measured in a luminometer
(Lumat LB 9507; Berthold) immediately after addition of the substrate
luciferin (Promega Co.).
-Galactosidase activity in the lysate was
measured using o-nitrophenyl-
-D-galactopyranoside as a
substrate, and the values were used to normalize the efficiency of
transfection. The mixture of plasmid DNA (0.5 µg) consisted of the
luciferase reporter vector (50 ng), an actin
5C-
-galactosidase reporter vector (50 ng), and 0.0-0.4 µg of
mutant Mblk-1 expression vector with 0.0-0.4 µg of empty pPac-PL
(total, 0.5 µg).
Expression and Purification of Glutathione S-transferase
(GST)-fused Proteins--
To prepare the truncated Mblk-1 mutants
(Mblk-1-(1-404), Mblk-1-(384-808), Mblk-1-(776-1207), or
Mblk-1-(1206-1597)), cDNA fragments corresponding to +275 to
+1486, +1424 to +2698, +2600 to +3895, and +3890 to +5065,
respectively, were amplified by PCR and subcloned into pET-22b
(Novagen, Inc., Madison, WI), whose MCS
(NdeI-XhoI) was replaced by His6-MCS-GST. Each
GST fusion protein was produced in Escherichia coli BL21
(DE3) and purified using glutathione-Sepharose 4B (Amersham
Biosciences).
In Vitro Phosphorylation Assay--
Reaction mixtures (30 µl)
contained Erk2, protein kinase A (PKA), and
Ca2+/calmodulin-dependent protein kinase II
(CaMKII) (New England Biolabs, Inc., Beverly, MA), 150 ng of each of
the truncated Mblk-1 protein, 100 or 200 µM ATP
containing [
-32P]ATP (1 µCi) in 50 mM
Tris-HCl (pH 7.5), and 10 mM MgCl2 for PKA, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
0.5 mM dithiothreitol, and 0.1 mM EDTA for
CaMKII, 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 0.1 mM EGTA, 2 mM
dithiothreitol, and 0.01% Brij 35 for Erk2. After incubation for 30 min at 30 °C, the reactions were terminated by adding SDS
sample buffer (150 mM Tris-HCl buffer, pH 6.8, containing 1.2% SDS, 30% glycerol, and 15% 2-mercaptoethanol), and then
subjected to SDS-polyacrylamide gel electrophoresis. The gel was then
fixed, dried, and subjected to autoradiography. Radioactivity was
measured by scanning the autoradiogram with a Bioimaging analyzer
(Fujifilm Co., Tokyo, Japan).
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RESULTS |
Mblk-1 Is a Sequence-specific Transcriptional Activator--
The
luciferase assay was used to examine whether Mblk-1 transactivates
genes containing MBEs in their promoters. Drosophila SL-2
cells were cotransfected with a luciferase reporter vector containing
MBEs and a minimal promoter and an Mblk-1 expression vector driven by
the Drosophila actin 5C promoter. Two kinds of minimal
promoters were used: the Hsp70Bb core promoter and the P-element core promoter with an hsp70 leader. With the
Hsp70Bb core promoter, Mblk-1 expression increased
luciferase activity ~2-fold depending on the MBE copy number (Fig.
1A). In contrast, neither the
transfection of an empty expression vector instead of the Mblk-1
expression vector nor the reporter vector containing UASGs
instead of MBEs increased the activity.

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Fig. 1.
Mblk-1 acts as a transcriptional activator in
transfected Drosophila SL-2 cells.
Drosophila SL-2 cells were transfected with 50 ng of
pGL3-basic vector containing the firefly luciferase gene, which is
under the control of Hsp70Bb core promoter (pGL3H)
(A) or P-element core promoter and hsp70
leader (pGL3PH) (B). Numbers below the
bars (0, 2, and 6) indicate
copy numbers of MBE or UAS that were subcloned upstream of the minimal
promoters. Cells were cotransfected with 50 ng of actin 5C
promoter- -galactosidase reporter vector as an internal control for
transfection efficiency and 0.4 µg of the Mblk-1 expression vector or
the empty vector. The error bars represent ±S.E. of
normalized luciferase activity from six independent
transfections.
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Using the P-element core promoter with the hsp70 leader,
Mblk-1 expression did not increase the activity for the reporter vector
containing two copies of MBE (Fig. 1B). Luciferase activity increased ~7-fold for the reporter vector containing six MBE copies as compared with that containing UASGs. This increase was
not observed when an empty vector was used instead of the Mblk-1
expression vector. These results indicate that Mblk-1 can transactivate
minimal promoters driven by MBE, but not those driven by
UASG. Furthermore, the level of transactivation correlated
with the MBE copy number. Thus, Mblk-1 is a sequence-specific
transcriptional activator.
Identification of the Functional Domains of Mblk-1--
To
identify the functional domains of Mblk-1, we created various deletion
mutants of Mblk-1 and examined their effects on the expression of the
reporter gene. Six series of N-terminal deletion mutants were first
constructed because this region contains some characteristic domains
such as 25-amino-acid residues that share high sequence homology (68%)
with CG18389/E93 (amino acid positions 29-53), Thr runs (106-130),
and Gln runs (164-177) (28). There were no significant differences
between the transcriptional activities of this series of deletion
mutants and wild-type Mblk-1 (Fig. 2A), indicating that
Mblk-1 function is independent of the specificity of the N-terminal 383 residues.
Another series of six deletion mutants was constructed to assess the
importance of the RHF1 and RHF2 domains. When the C-terminal 390 amino
acid residues were deleted (
1208-1597), there was no appreciable
effect (Fig. 2B). When the
C-terminal 399 residues including the RHF2 domain (
809-1597) were
deleted, however, the transcriptional activity of Mblk-1 was almost
completely lost. In contrast, the luciferase activity gradually
decreased as the N-terminal regions were deleted (
1-383,
1-775). In addition, deletion of the 430 residues including the
RHF2 domain (
1205-1597) caused complete loss of transcriptional
activity (Fig. 2B). These results indicate that the 399 residues including the RHF2 domain are necessary for Mblk-1
function.

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Fig. 2.
Effects of deletion mutants of Mblk-1 on the
expression of the reporter gene. A series of N-terminal deletion
mutants (A), N-terminal and C-terminal deletion mutants
(B), and internal deletion mutants of Mblk-1 (C)
were assayed using the luciferase assay. The upper part of each panel shows a schematic
representation of the structures of the deletion mutants of Mblk-1. The
lower part represents the relative luciferase activity of
SL-2 cells transfected with 0.4 µg of the expression vector for each
mutant Mblk-1. The number of amino acid residues deleted in each mutant
is shown at the left of each bar. Cells were
cotransfected with 50 ng of actin 5C
promoter- -galactosidase reporter vector and 50 ng of the pGL3PH
containing six tandem copies of MBE. The error bars
represent ±S.E. of normalized luciferase activity from six independent
transfections.
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To directly assess the significance of RHF1 and RHF2 for Mblk-1
transcriptional activity, RHF1 and RHF2 deletion mutants of Mblk-1 were
created and examined using the luciferase assay (Fig. 2C).
The transactivation activity of the RHF2 deletion mutant (
1031-1088) was almost completely lost. In contrast, RHF1
(
586-636) deletion did not have an appreciable inhibitory effect,
indicating that the RHF2 domain is necessary for Mblk-1 transcriptional
activity (Fig. 2C). In contrast, RHF1 is dispensable, at
least in this assay system.
MAPK Phosphorylates Mblk-1 at Ser-444--
Recent work established
the crucial role of second messenger-dependent kinase(s)
such as PKA, CaMKII, and MAPK in the modulation of neuronal activity
and their involvement in learning and memory (29-32). In the honeybee,
PKA and CaMKII are strongly expressed in the MBs (33, 34). These
findings led us to hypothesize that one or some
signal-dependent kinase(s) regulates Mblk-1 activity by
phosphorylation. To test this hypothesis, we first examined whether
Mblk-1 can be phosphorylated by these kinases in vitro. For
this, four different truncated Mblk-1 proteins were expressed separately as GST fusion proteins (GST-Mblk-1-(1-404),
GST-Mblk-1-(384-808), GST-Mblk-1-(776-1207), and
GST-Mblk-1-(1206-1597)) and tested as substrates in protein kinase
assays (Fig. 3A).

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Fig. 3.
Phosphorylation of Mblk-1 in
vitro. A, schematic representation of
truncated GST-Mblk-1 fusion proteins. B, phosphorylation of
the truncated Mblk-1 in vitro. The recombinant Mblk-1
proteins were incubated for 30 min at 30 °C with ERK2, PKA, or
CaMKII in the presence of [ -32P]ATP followed
by SDS-polyacrylamide gel electrophoresis, and then the gel
was fixed, dried, and subjected to autoradiography. Radioactivity was
measured by scanning the autoradiogram.
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Three of the four truncated Mblk-1 mutants were significantly
phosphorylated by one of these kinases in vitro:
GST-Mblk-1-(1-404) and GST-Mblk-1-(1206-1597) were preferentially
phosphorylated by CaMKII, whereas GST-Mblk-1-(384-808) was
preferentially phosphorylated by ERK2 and PKA (Fig. 3B).
There was no significant phosphorylation when GST-Mblk-1-(776-1207)
was used as a substrate. Consistently, Mblk-1 contains consensus
recognition/phosphorylation sequences for the MAPK (amino acid
positions 442-445), PKA (amino acid positions 412-415), and CaMKII
(amino acid positions 203-206, 293-296, 412-415, 1064-1067,
1267-1270, 1355-1358, 1393-1396, 1405-1408) substrates (35,
36).
Among them, we focused on the MAPK consensus phosphorylation site
(Ser-444) as there was only a single site in Mblk-1 and the
phosphorylation by MAPK was relatively clear. To examine whether Ser-444 was actually phosphorylated by MAPK, we mutated the putative phospho-acceptor residue, Ser-444, in the GST-Mblk-1-(384-808) to Ala
(Fig. 4A) and tested the
mutated substrate in the protein kinase assay. The mutant protein
(GST-Mblk-1-(384-808)S444A) was not phosphorylated by MAPK (Fig.
4B). The results indicated that Mblk-1 can be directly
phosphorylated at Ser-444 by MAPK in vitro.

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Fig. 4.
Phosphorylation of Mblk-1-(384-808) and
Mblk-1-(384-808)S444A by MAPK. A, schematic
representation of GST-Mblk-1-(384-808) and GST-Mblk-1-(384-808)S444A
proteins. The location of the single MAPK consensus
phosphorylation site (PX(S/T)P) in Mblk-1 is indicated by
single-letter amino acid codes. The phosphorylated or mutant residue is
underlined. B, phosphorylation of the truncated
Mblk-1 by MAPK in vitro. The recombinant Mblk-1 proteins
were incubated for 30 min at 30 °C with ERK2 in the presence of
[ -32P]ATP followed by SDS-polyacrylamide gel
electrophoresis, and then the gel was fixed, dried, and subjected to
autoradiography. Radioactivity was measured by scanning the
autoradiogram.
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Mblk-1-induced Transactivation Was Stimulated by the
Ras/MAPK Pathway--
We next tested whether
phosphorylation at Ser-444 by MAPK affected Mblk-1 transcriptional
activity. The increase in luciferase activity was reduced to ~65%
when the Mblk-1S444A protein was expressed instead of intact Mblk-1
(Fig. 5A). These results
strongly suggest that the activity of Mblk-1 can be modulated, at least in part, by direct phosphorylation by MAPK. To test this possibility further, we examined whether the Ras/MAPK pathway stimulated Mblk-1 transcriptional activity. For this, either the pPacMAPKSem
or the pPacRas1V12 plasmid, which express an activated form
of Drosophila MAPK (37) or Ras1 (38, 39), respectively, was
cotransfected, and luciferase activity was examined. Cotransfection of
pPacMAPKSem or pPacRas1V12 increased the
transcriptional activity of intact Mblk-1 ~2-fold (Fig.
5B). In contrast, neither MAPKSem nor
Ras1V12 had any effect on basal activation. These results
clearly indicated that Mblk-1-induced transactivation can be stimulated
by the Ras/MAPK pathway. Mblk-1S444A transcriptional activity, however,
was also increased to some extent by expression of activated Ras1 or
MAPK, suggesting that Mblk-1 was also modulated by the Ras/MAPK pathway at a site other than Ser-444.

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Fig. 5.
The effect of the expression of the
active-form Ras1 and MAPK on Mblk-1 and Mblk-1S444A activity.
A, relative luciferase activity of SL-2 cells transfected
with 0.4 µg of the expression vector for Mblk-1 or Mblk-1S444A. *,
p < 0.05. B, analysis of the stimulation of
Mblk-1 or Mblk-1S444A by activated Ras1 or MAPK. SL-2 cells were
cotransfected with 0.2 µg of the expression vector for Mblk-1 or
Mblk-1S444A as well as 0.2 µg of the expression vector for
Ras1V12 or MAPKSem. Cells were cotransfected
with 50 ng of actin 5C- -galactosidase reporter vector and
50 ng of the pGL3PH containing six tandem copies of MBE. All
transfections were adjusted to 0.5 µg of total DNA with pPac-PL. The
error bars represent ±S.E. of normalized luciferase
activity from six independent transfections.
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Treatment with forskolin induces activation of endogenous PKA in SL-2
cells (40). Thus, we also examined the effect of PKA activation by
treatment with forskolin on Mblk-1transcriptional activity. There was
no detectable effect, however, on Mblk-1 transactivation (data not
shown), suggesting that PKA has little effect on Mblk-1 activation,
although PKA phosphorylated Mblk-1 in vitro.
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DISCUSSION |
We previously identified the preferred binding sequence of Mblk-1,
termed MBE. It remained uncertain, however, whether Mblk-1 has
transcriptional activity and whether MBE is important for Mblk-1-mediated transcriptional activity. We report the first direct
evidence for the transcriptional activity of Mblk-1/E93 insect
proteins. Thus, the honeybee Mblk-1 is the first transcription factor
identified that is expressed preferentially in the MBs of the insect brain.
Deletion experiments revealed that Mblk-1 contains functional regions
for activation. Among them, RHF2 was necessary for Mblk-1 activity.
Although truncated Mblk-1 containing RHF1 can also bind to MBE in
vitro (13), there was no appreciable decrease in the transactivation activity of RHF1 deletion mutants in the luciferase assay. At present, it is not clear whether RHF1 has some functions in
Mblk-1. It is possible, however, that truncated RHF1 binds to MBE
in vitro because the domain is readily exposed to bind to
MBE, whereas RHF1 is usually hidden in the intact Mblk-1 molecule, to
be exposed only in response to a particular signal(s). Our luciferase
assay might have lacked such a particular signal(s) and thus failed to
detect any effect of RHF1 deletion.
Covalent modification by phosphorylation is a potential route for
Mblk-1 regulation. Previous studies established a crucial role of
second messenger-dependent kinases in the modulation of neuronal activity, and their involvement in learning and memory (29-32) and many different types of stimuli that affect gene
expression also leads to the activation of protein kinases (41). Thus, it is likely that Mblk-1 function is also regulated by phosphorylation. We demonstrated that Mblk-1 activity could be modulated by direct phosphorylation by the Ras/MAPK pathway. Specifically, we identified Ser-444 as one of the important phosphorylation sites involved in
determining the magnitude of the Mblk-1 transactivating capacity. It
remains unknown, however, how the activity of Mblk-1 can be stimulated
by the phosphorylation by MAPK. We previously reported that Mblk-1
functions as a dimer using an in vitro pull-down assay (13).
Phosphorylation did not have a significant effect, however, on
homophilic protein-protein interactions (data not shown). Some noteworthy possibilities are: 1) phosphorylation allows translocation of Mblk-1 into the nucleus (42), 2) DNA binding activity of Mblk-1
might be modulated by phosphorylation (43), and 3) phosphorylation might affect interaction of the transactivation domains with the transcriptional machinery (44, 45).
Long term memory formation is generally dependent on protein synthesis
(14), and a role for MAPK in long term memory has been demonstrated in
a number of different learning paradigms in invertebrates and
vertebrates (17-23, 32). Among the transcription factors involved in
learning, memory, and neuronal plasticity, cAMP-response
element-binding protein (CREB) is best characterized (46, 47). CREB
transcriptional activity is also stimulated by the Ras/MAPK pathway,
and the Ras/MAPK-dependent phosphorylation of CREB is
performed by several different kinases, including members of the
ribosomal S6 kinase and mitogen- and stress-activated protein kinase
families (48-50). Furthermore, CREB is also activated via PKA and CaMK
pathways (51, 52). Similarly, the phosphorylation of Mblk-1 by PKA and
CaMKII in vitro (Fig. 3B) and the partial increase in the transcriptional activity of Mblk-1S444A by the Ras/MAPK
pathway (Fig. 5B) suggest that Mblk-1 is modulated via various signaling pathways other than the Ras/MAPK pathway.
We previously demonstrated that gene expression for inositol
1,4,5-triphosphate (IP3) receptor, CaMKII, and
IP3 phosphatase is concentrated in the large-type Kenyon
cells of the honeybee brain (34, 53, 54). PKA is also expressed
preferentially in the large-type Kenyon cells (33). To our knowledge,
Mblk-1 is the first MB-selective transcription factor that might
participate in transcriptional activation of some genes for proteins
involved in synaptic plasticity like IP3 receptor,
CaMKII, PKA, and IP3 phosphatase in the MB neural circuits
and might therefore be responsible for the status of MBs as the main
association and memory centers of the honeybee brain. The
identification of possible target genes for Mblk-1 and its biologic
function might provide important clues to the molecular basis that
underlies the MB functions.