The Activity of Mblk-1, a Mushroom Body-selective Transcription Factor from the Honeybee, Is Modulated by the Ras/MAPK Pathway*

Jung-Min Park, Takekazu Kunieda, and Takeo KuboDagger

From the Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, January 16, 2003, and in revised form, March 7, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We previously identified a gene, termed Mblk-1, that encodes a putative transcription factor with two DNA-binding motifs expressed preferentially in the mushroom body of the honeybee brain, and its preferred binding sequence, termed Mblk-1-binding element (MBE) (Takeuchi, H., Kage, E., Sawata, M., Kamikouchi, A., Ohashi, K., Ohara, M., Fujiyuki, T., Kunieda, T., Sekimizu, K., Natori, S., and Kubo, T. (2001) Insect Mol Biol 10, 487-494; Park, J.-M., Kunieda. T., Takeuchi, H., and Kubo, T. (2002) Biochem. Biophys. Res. Commun. 291, 23-28). In the present study, the effect of Mblk-1 on transcription of genes containing MBE in Drosophila Schneider's Line 2 cells was examined using a luciferase assay. Mblk-1 expression transactivated promoters containing MBEs ~2-7-fold. Deletion experiments revealed that RHF2, the second DNA-binding domain of Mblk-1, was necessary for the transcriptional activity. Furthermore, mitogen-activated protein kinase (MAPK) phosphorylated Mblk-1 at Ser-444 in vitro, and the Mblk-1-induced transactivation was stimulated by phosphorylation of Ser-444 by the Ras/MAPK pathway in the luciferase assay. These results suggest that Mblk-1 is a transcription factor that might function in the mushroom body neuronal circuits downstream of the Ras/MAPK pathway in the honeybee brain.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta 1-51, Delta 1-99, and Delta 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 (Delta 1-51, Delta 1-99, and Delta 1-142/pPac-PL, respectively). To create two other N-terminal deletion mutants (Delta 1-220 and Delta 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 (Delta 411-1597), +1424 to +2698, +2600 to +3895, and +3890 to +5065 (Delta 1-1204), respectively, were amplified by PCR. A cDNA fragment corresponding to +1424 to +2698 was used to replace the NotI-SpeI fragment of Delta 411-1597, and the resulting insert was subcloned into pPac-PL (Delta 809-1597/pPac-PL). A cDNA fragment corresponding to +2600 to +3895 was used to replace the BlpI-SpeI fragment of Delta 809-1597, and the resulting insert was subcloned into pPac-PL (Delta 1208-1597/pPac-PL). A cDNA fragment corresponding to +2600 to +3895 was used to replace the BamHI-NcoI fragment of Delta 1-1204, and the resulting insert was subcloned into pPac-PL (Delta 1-775/pPac-PL). A cDNA fragment corresponding to+1424 to +2698 was used to replace the BamHI-BlpI fragment of Delta 1-775, and the resulting insert was subcloned into pPac-PL (Delta 1-383/pPac-PL).

To create the RHF1 deletion mutant (Delta 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 (Delta 586-636/pPac-PL). To create the RHF2 deletion mutant (Delta 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 (Delta 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.). beta -Galactosidase activity in the lysate was measured using o-nitrophenyl-beta -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-beta -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 [gamma -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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

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 (Delta 1208-1597), there was no appreciable effect (Fig. 2B). When the C-terminal 399 residues including the RHF2 domain (Delta 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 (Delta 1-383, Delta 1-775). In addition, deletion of the 430 residues including the RHF2 domain (Delta 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-beta -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.

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 (Delta 1031-1088) was almost completely lost. In contrast, RHF1 (Delta 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 [gamma -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.

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 [gamma -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.

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-beta -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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

We thank Dr. Tien Hsu (Medical University of South Carolina) for providing the pPacRas1V12 and pPacMAPKSem plasmids. We are also grateful to Dr. Hideaki Takeuchi (The University of Tokyo) for the kind encouragement during this work and to Dr. Naohiko Aozasa (Kanazawa University) and Dr. Yukiko Gotoh (The University of Tokyo) for valuable discussion.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-3-5841-4446; Fax: 81-3-5800-3553; E-mail: stkubo@mail.ecc.u-tokyo.ac.jp.

Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M300486200

    ABBREVIATIONS

The abbreviations used are: MB, mushroom body; MBE, Mblk-1-binding element; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CREB, cAMP-response element-binding protein; GST, glutathione S-transferase; MCS, multicloning site; IP3, inositol 1,4,5-triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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