Insect Basic Leucine Zipper Proteins and Their Role in Cyclic AMP-Dependent Regulation of Gene Expression

Insect Basic Leucine Zipper Proteins and Their Role in Cyclic AMP-Dependent Regulation of Gene Expression Jeroen Poels and Jozef Vanden Broeck Laboratory for Developmental Physiology, Genomics and Proteomics, Catholic University Leuven, B-3000 Leuven, Belgium

The cAMP-protein kinase A (PKA) pathway is an important intracellular signal transduction cascade that can be activated by a large variety of stimuli. Activation or inhibition of this pathway will ultimately affect the transcriptional regulation of various genes through distinct responsive sites. In vertebrates, the bestcharacterized nuclear targets of PKA are the cyclic AMP response element-binding (CREB) proteins. It is now well established that CREB is not only regulated by PKA, but many other kinases can exert an effect as well. Since CREB-like proteins were also discovered in invertebrates, several studies unraveling their physiological functions in this category of metazoans have been performed. This review will mainly focus on the presence and regulation of CREB proteins in insects. Differences in transcriptional responses to the PKA pathway and other CREB-regulating stimuli between cells, tissues, and even organisms can be partially attributed to the presence of different CREB isoforms. In addition, the regulation of CREB appears to show some important differences between insects and vertebrates. Since CREB is a basic leucine zipper (bZip) protein, other insect members of this important family of transcriptional regulators will be briefly discussed as well. KEY WORDS: Insect, CREB, Basic leucine zipper (bZip), Transcription, Splicing, Kinase, cAMP, Calcium (Ca2þ), Memory. ß 2004 Elsevier Inc.

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I. Introduction Metazoans contain hundreds of diVerent cell types, each designed to perform a specific role that contributes to the overall functioning of the organism. Every one of these cells contains the same gene set. Specialization of particular cells is achieved through the tightly controlled expression and regulation of a precise subset of genes. In addition, the gene expression pattern of cells is dynamic in that it can respond to physiological and environmental stimuli, a feature that is particularly important in development. The expression level of most genes is regulated by transcription factors that bind to regulatory DNA sequences upstream of the transcription initiation site. The fact that more than 5% of the human genome possibly encodes transcription factors emphasizes the importance of this functional class of proteins (Orphanides and Reinberg, 2002; Tupler et al., 2001). The activities of these transcription factors are controlled by a diverse array of regulatory pathways. Molecular studies have uncovered a remarkable conservation of specific transcriptionregulating proteins between vertebrates and invertebrates. This review will focus on one of the earliest identified and most widely expressed stimulusinduced transcription factors, i.e., the cAMP response element-binding protein (CREB). More specifically, it will deal with the occurrence, the regulation, and the physiological function of CREB and CREB-related proteins in insects. In addition, the superfamily to which CREB proteins belong, the basic leucine zipper proteins, will also be discussed.

II. Basic Leucine Zipper Proteins A. The Basic Leucine Zipper Domain One important group of signal-regulated transcription factors constitutes the basic leucine zipper (bZip) proteins. Members of this family all contain a C-terminal basic domain that mediates binding to the major groove of sequence-specific double-stranded DNA and a leucine zipper domain that facilitates dimerization (Landschulz et al., 1988; Vinson et al., 1989). A schematic representation of bZip proteins, attached to their DNA recognition sequences, is given in Fig. 1. As demonstrated by X-ray crystal studies, the leucine zipper domain is a dimeric parallel coiled-coil, typically composed of four to five heptad repeats of amino acids (Ellenberger et al., 1992; O’Shea et al., 1991). The amino acids in the heptad are labeled using the nomenclature (a,b,c,d,e,f,g)n. The a and d positions usually contain hydrophobic amino acids (with leucine typically in the d position), and are on the same side of the a-helix, composing a hydrophobic interface that contributes to

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FIG. 1 X-ray structure of GCN4 bZip motif bound to a TRE DNA sequence. The DNA is in red, while the bZip a-helices are in blue with the leucines in the ‘‘d’’ position shown in gray (Ellenberger et al., 1992; Fassler et al., 2002). [Reprinted, with permission, from Fassler et al. (2002). Genome Res. 12, 1190–1200. #2002 by Cold Spring Harbor Laboratory Press.]

dimerization stability. The e and g positions are more exposed and often contain charged and polar amino acids. The coiled-coil regions are used by the bZip proteins to homo- and heterodimerize with each other, bringing together the two clusters of basic amino acids in such a way that they can bind to a dyad-symmetric DNA-binding site. Heterodimerization can therefore change the DNA-binding properties of a dimeric complex (Hai and Curran, 1991). Interestingly, bZip proteins display a high degree of partnering selectivity, which allows them to function in diverse pathways. Recently, methods for predicting possible dimerization partners between bZip proteins are becoming more and more reliable tools that can be used to screen the existing finished genome sequencing projects (Fassler et al., 2002; Fong et al., 2004). The number of predicted bZip proteins in the human genome is 65, while 31 were identified in Caenorhabditis elegans and 27 members were found in Drosophila melanogaster. The most studied members of this superfamily are the activating protein-1 (AP-1; Jun/Fos) and CRE-binding protein/activating transcription factor (CREB/ATF) proteins that bind to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE, consensus sequence TGACTCA) and cAMP response element (CRE, consensus sequence TGACGTCA), respectively (Karin and Smeal, 1992). AP-1 is regarded as a nuclear messenger that mediates the actions of signal transduction pathways stimulated by growth factors, hormones, neurotransmitters, cytokines, and cellular stresses. The CREB/ATF proteins are stimulated via the protein kinase A (PKA) pathway, but many other kinases were later shown to be involved in CREB/ATF regulation (Bito et al., 1996; Dash et al., 1991; de Groot et al., 1994; Ginty et al., 1994; Gonzalez and Montminy,

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1989; Kimura et al., 2002; Matthews et al., 1994; Sheng et al., 1991; Xing et al., 1996; Yamamoto et al., 1988). CRE-binding proteins will be further discussed below.

B. bZip Proteins in Drosophila melanogaster A genome-wide analysis revealed 29 bZip proteins in Drosophila melanogaster (Rubin et al., 2000), a number that was later refined to 27 (Fassler et al., 2002). Approximately half of these bZip proteins have been isolated (Table I), with kayak (kay) and Jun-related antigen (Jra or DJun) as the first two to be isolated (Perkins et al., 1988, 1990; Zhang et al., 1990). Kayak got its name from the prominent dorsal hole that mutants in this gene display, making preparations of the cuticle look like a small boat. The gene is the Drosophila homolog of mammalian Fos and thus also termed DFos or Fos-related antigen (Fra). Only single gene homologues of c-Fos and c-Jun have been identified in Drosophila, making this organism an excellent study object for AP-1 function. DJun and DFos can either form homo- or heterodimers in Drosophila, whereas in mammals Fos homodimers do not form in physiological conditions. Both bZip proteins are required for dorsal closure during embryogenesis (Ciapponi and Bohmann, 2002; Riesgo-Escovar and Hafen, 1997a,b; Zeitlinger et al., 1997). This process is typified by the dorsalward stretching of the lateral epidermis over the amnioserosa (an embryonic cell layer that disintegrates later on) from both sides. Eventually, this leads to the enclosure of the embryo with epidermis. In addition to DJun and DFos, mutations in Jun NH2-terminal kinase (DJNK) and DJNK kinase (DJNKK) also show a defect in dorsal closure. A downstream target of activated DJun and DFos during dorsal closure is decapentaplegic (dpp). In a reciprocal manner, DFos expression is dependent on Dpp function in cells of the lateral epithelium. Similarly, in late embryogenesis, Dpp that originates from the overlaying visceral mesoderm regulates DFos expression in the endoderm. Dpp plays a key role during endoderm induction, whereby it stimulates transcription of the homeotic gene labial. As a result, diVerent cell types of the larval gut are specified (Eresh et al., 1997; Riese et al., 1997). Semilethal hypomorphic alleles of some of the genes that are required for dorsal closure result in adults that display a split thorax. The dorsal thorax is made during metamorphosis from the proximal portions of the left and right wing imaginal discs that fuse 6–8 h after pupariation. Thus, DFos and the JNK pathway are not only required in embryonic dorsal closure, but also for fusion of the imaginal discs during pupal development (Zeitlinger and Bohmann, 1999). A separate role for DJun can be found in Drosophila eye development (Bohmann et al., 1994). Each of the 800 single eye units (ommatidia) of the

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BASIC LEUCINE ZIPPER PROTEINS IN INSECTS TABLE I Members of the bZip Gene Family Isolated from Drosophila melanogaster a

Gene

Mammalian counterpart (Fassler et al., 2002)

Leucine zipper structure

Biological functions/ involved processes

DJun

JUND

LxLxLxLxL

Eye development, dorsal closure, NMJ plasticity

DFos

FOS

LxLxMxLxL

Dorsal and thorax closure, NMJ plasticity

Slow border cells

C/EBP

LxLxMxHxL

Oogenesis

Cap-n-collar

NF-E2

LxVxLxLxIxL

Development of labral and mandibular structures, oogenesis

Giant

HLF

LxLxLxF

Terminal region determination

Sisterless-a

None

LxLxIxL

Sex determination, gut development

Vrille

NF-IL3

LxLxLxI

Circadian rhythm, regulation of the actin cytoskeleton

PAR-domain protein 1

HLF

LxLxLxL

Circadian rhythm

Apontic

None

LxLxLxL

Trachea, heart, and head development, oogenesis

A3-3

ATF-3

LxLxLxLxL

Oogenesis

Cryptocephal

ATF-4

LxLxRxL

Ecdysone response regulation

a bZip genes isolated from Drosophila (column 1), their mammalian homologue (column 2), and leucine zipper structure given in single character amino acid code, where ‘‘x’’ represents a stretch of six amino acids (column 3). Column 4 indicates the biological function or the physiological processes in which the respective bZip protein is involved, as discussed in the text.

Drosophila eye contains a stereotypic arrangement of eight photoreceptor cells (R1–R8). During late larval and pupal stages the eye develops progressively from a monolayer epithelium. The fates of the photoreceptor cells are determined exclusively by inductive interactions between neuronal precursors in the cell cluster from which the ommatidium is formed. R8 is the first cell to diVerentiate, while R7 is recruited last. The development of the R7 photoreceptor cell is determined by a specific inductive interaction with the R8 photoreceptor cell. This process is mediated by the bride of sevenless, BOSS, a cell-surface-bound ‘‘ligand,’’ and the sevenless receptor tyrosine kinase (SEV). The BOSS ‘‘ligand,’’ a seven transmembrane protein of 896 amino acids, is expressed on the surface of the developing R8 cell (Hart et al.,

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1990; Raabe, 2000; Van Vactor et al., 1991). Without SEV or BOSS, the R7 precursor cell will develop into a nonneuronal cone cell (Reinke and Zipursky, 1988; Tomlinson and Ready, 1986). The immediate consequence of the BOSS–SEV interaction is the stimulation of SEV kinase activity and autophosphorylation on tyrosine residues. A major route by which SEV, as well as receptor tyrosine kinases in general, transduces signals involves activation of the RAS-MAPK pathway. At the end of this cascade stands Rolled, a serine/threonine protein kinase that possesses DJun as one of its nuclear targets. Expression of a DJun transgene that lacks phosphoacceptor sites suppresses the diVerentiation of photoreceptor cells, while a mutant in which the phosphorylation sites are replaced by phosphatemimetic Asp residues can promote photoreceptor diVerentiation (Peverali et al., 1996). In Drosophila, 6–10 follicle cells, the border cells, migrate through the developing egg chamber during oogenesis. By P element insertion mutations, a locus was defined and termed slow border cells (slbo), because hypomorphic alleles caused delayed onset of the migration. The slbo locus encodes a bZip protein homologous to mammalian CCAAT/enhancer-binding protein (C/EBP) (Montell et al., 1992; Rorth and Montell, 1992). Downstream targets for slbo include shotgun, a Drosophila homologue of E-cadherin, a cell–cell adhesion molecule that is upregulated during migration of border cells. Shotgun is required for migration, whereby the expression level determines the migration speed (Niewiadomska et al., 1999). Another downstream target is the breathless gene (btl), which is expressed in cells of the developing tracheal system and encodes a protein similar to mammalian fibroblast growth factor (FGF) receptor (Murphy et al., 1995). JING, a nuclear protein containing three zinc finger motifs, is expressed in border cells depending upon slbo. The mutant phenotype associated with hypomorphic slbo alleles can be rescued by induced jing expression. JING is likely to cooperate with SLBO in activating transcription from downstream targets (Liu and Montell, 2001). Another locus that was found to independently cause border cell migration defects is taiman (tai), encoding a protein similar to the steroid receptor coactivator protein amplified in breast cancer 1 (AIB1). TAI interacts with the ecdysone receptor complex, suggesting that ecdysone can regulate border cell migration (Bai et al., 2000). Cap-n-collar (cnc) was originally proposed to form a heterodimeric regulatory protein involved in the control of head morphogenesis (Mohler et al., 1991). It was named based on its expression pattern in the anteriormost labral segment (cap) and the mandibular segment (collar) of embryos. Cnc functions are required for normal development of labral and mandibular structures (Mohler et al., 1995). Three diVerent isoforms are encoded by cnc (CncA, B, and C). All three forms contain a bZip domain, but diVer in the length of their N-terminal parts, with CncC being the longest and CncA

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being the shortest protein. The CncB protein possesses properties that allow it to selectively repress many regulatory elements that are activated by the homeotic gene Deformed. It has been suggested that Deformed requires the action of the cnc gene product to elicit a mandibular fate whereas without cnc, it determines maxillary structures. Mutant embryos with reduced levels of the CncB isoform contain a duplicated set of mouth hooks and cirri instead of mandibular structures (McGinnis et al., 1998). Double-stranded RNA interference experiments have shown that CncA and CncC are dispensable for embryonic development (Veraksa et al., 2000). Recently, the CncC isoform was found to be required for stable anchoring of the oocyte nucleus during Drosophila oogenesis (Guichet et al., 2001; Ruden and Zhang, 2003). The Giant gene (gt), which was cloned in 1992, belongs to the gap genes, so called because mutants in this class of genes have gaps in their structures. Gt encodes a transcriptional repressor (Capovilla et al., 1992). Lack of gt expression causes gaps in both anterior and posterior structures, specifically the labial and labral structures and abdominal segments A5 through A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Giant regulates the expression of gap and pair-rule genes, including even-skipped, hunchback, and Kru¨ppel (Capovilla et al., 1992; Eldon and Pirrotta, 1991; Kraut and Levine, 1991a,b; Stanojevic et al., 1991; Wu et al., 1998, 2001). A functional interaction of giant with the abdominal-A homeotic gene has also been established, whereby the regulatory elements from the abdominal-A gene contain binding sites for giant (Shimell et al., 2000; Strunk et al., 2001). Giant represses expression over a short distance (100–150 bp) enabling the protein to repress the activity of one enhancer, while not interfering with another (Hewitt et al., 1999). Depending on the promoter context, giant-mediated repression can occur via interaction with the C-terminal-binding protein, a corepressor that interacts with other short-range repressors, such as Kru¨ ppel (Nibu and Levine, 2001; Nibu et al., 1998a,b; Strunk et al., 2001). In the developing fly, sexual identity is determined by zygotic X chromosome dose, i.e., the ratio of X chromosomes to sets of autosomes (X:A). In females, where two X chromosomes are present, the ratio of X-linked genes (ratio of 1, i.e., 2X:2A) is higher than in males (ratio of 0.5, i.e., 1X:2A) where the Y chromosome does not possess a sex-determining function. Sisterless-a (sisA) is one of a number of factors on the X chromosome responsible for sex determination (Cline, 1988; Deshpande et al., 1995; DuVy and Gergen, 1991; Erickson and Cline, 1993; Liu and Belote, 1995; Steinmann-Zwicky, 1993). The master gene in sex determination is Sex lethal (Sxl), whose expression is being repressed in males and activated in females (Bopp et al., 1991;

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Parkhurst et al., 1990). Sisterless-A is one of the transcription factors that bind the promoter region of Sxl and enhance transcription (Erickson and Cline, 1993, 1998; Estes et al., 1995). This induction does not occur in males who have only one X chromosome (Cline and Meyer, 1996; Schutt and Nothiger, 2000). Sex lethal is an RNA splicing enzyme with Transformer mRNA as a direct target, acting positively on the functional splicing of Transformer mRNA. Transformer, by itself a splice factor, is acting in turn on downstream RNAs that require sex-specific splicing in the female Drosophila flies (Sosnowski et al., 1994). Drosophila melanogaster SisterlessA contains a rather unusual bZip domain. As an example, the residue in the ‘‘a’’ position of the zipper region, which generally contains a hydrophobic amino acid, is formed by A-R-E-G in the four heptad repeats. This feature, as well as other sex-determining mechanisms, is conserved across the genus of Drosophila (Erickson and Cline, 1998). The R and A residues likely prevent homodimerization, but a possible partner would be cryptocephal (Fassler et al., 2002). SisA expression was also found to be high in yolk nuclei of the embryo suggesting a second role, apart from sex determination, for this bZip protein (Erickson and Cline, 1993, 1998), a hypothesis that was later confirmed in a study using diVerent sisA alleles (Walker et al., 2000). The vrille (vri) gene was originally isolated due to its role in the decapentaplegic developmental pathway (George and Terracol, 1997). It is also involved in normal hair and cell growth and proliferation via the regulation of the actin cytoskeleton (Szuplewski et al., 2003). Furthermore, it is characterized as a clock-controlled gene that is expressed in circadian pacemaker cells and acts as a transcriptional repressor of the Clock (Clk) gene (Blau and Young, 1999; Cyran et al., 2003; Glossop et al., 2003). PAR-domain protein 1 (Pdp1), a protein that belongs to the PAR family of bZip proteins, is also a regulator of circadian rhythms and is located in the same regulatory feedback loop as VRI (Cyran et al., 2003; Lin et al., 1997; Reddy et al., 2000). The use of alternative promoters and alternative splicing generates six Pdp1 isoforms in vivo. VRI and Pdp1 have opposite functions in the Drosophila clock; both transcription factors compete for the same regulatory sites in the Clk promoter, whereby VRI and Pdp1 serve as a repressor and an activator of Clk transcription respectively. Transcription of both bZip proteins is activated by the dCLOCK/CYCLE (dCLK/CYC) heterodimer, but diVerent phases of vri and Pdp1 RNA and protein accumulation separate the times at which dClk expression is repressed or activated (Cyran et al., 2003; McDonald and Rosbash, 2001). Pdp1 was originally isolated and characterized as a component of the muscle activator region, where it is involved in regulating expression of the Tropomyosin I gene (Lin et al., 1997). The apontic gene, also termed trachea defective (tdf), encodes a putative transcriptional regulator that enables cells to migrate during tracheal branch outgrowth and tube formation (the respiratory system of the fly) (Eulenberg

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and Schuh, 1997). Furthermore, Apontic is also required for the morphogenesis and function of the Drosophila heart (Su et al., 1999) and for head development where it is required for the elaboration of dorsal and ventral head structures (Gellon et al., 1997). A fourth function was discovered in 1999 (Lie and Macdonald, 1999), showing another aspect of the multiple biological roles that this protein displays. Apontic acts together with Bruno (an mRNA-binding protein) in translational repression of Oskar mRNA. The product of the Oskar gene serves two functions: determination of germ cell fate and determination of posterior polarity in the oocyte. This adds a role of Apt in Drosophila oogenesis. Thus, Apontic is not always located in the nucleus, but can exert a function as a repressor of mRNA translation in the cytosol of cells where it is expressed. In addition, the basic domain of this putative bZip protein is rather unusual in that it resides very close to the leucine zipper and that it contains fewer basic residues than most bZip proteins. Recently, a coactivator of Apontic was identified as multiprotein bridging factor 1 (MBF1), a cofactor that was originally shown to interact with the nuclear receptor FTZ-F1 in the silk moth Bombyx mori (Li et al., 1994). MBF1 forms a ternary complex with TATA-binding protein (TBP) and Apontic and is necessary for Apontic function (Liu et al., 2003). The gene A3-3 encodes a bZip protein with similarity to ATF-3. One mutation was described whereby homozygotes showed reduced viability during larval development. Mutant females mated to wild-type males laid eggs, but the majority of these do not show any development. The percentage of defective eggs increased with the age of the female flies (Heitzeberg et al., 1999). Although already described in 1946 (Hadorn and Gloor, 1946), the cryptocephal gene (crc) was not cloned until more than 50 years later (Hewes et al., 2000). Crc was named for the mutant phenotype that displays defective eversion of the adult head. Phenotypic parallels between the crc mutant phenotype (Chadfield and Sparrow, 1985; Sparrow and Chadfield, 1982) and mutations in several ecdysone response genes (Broadus et al., 1999; D’Avino and Thummel, 1998; Fletcher et al., 1995; Kiss et al., 1988; Lam et al., 1999) indicate an involvement in common regulatory pathways. Crc encodes at least six mRNA and three protein isoforms (CRC-A, CRC-B, and CRC-D). While the CRC-A and CRC-B isoform diVer only at their N-terminus, CRC-D is a much shorter (79 amino acids in comparison to 373 for CRC-A) C-terminally truncated form of CRC-A that does not contain a bZip domain (Hewes et al., 2000). CRC-D could therefore function as a dominant negative regulator by competing with CRC-A for proteinbinding sites. Crc mutant alleles define multiple roles for the isoforms during development, whereby CRC-A and/or CRC-B are implicated in larval molting, e.g., by functioning in ecdysone biosynthesis. In conclusion, crc has a central role in the regulation of ecdysone biosynthesis/secretion or in determining the responses of target tissues to steroids (Hewes et al., 2000).

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III. cAMP Response Element Binding (CREB) Proteins A. An Introduction to CREB Many genes that are transcriptionally regulated by cAMP contain one or more conserved sequences in their 50 flanking region that are identical or similar to the palindromic octamer 50 -TGACGTCA-30 . Hence, this sequence, which was first discovered in the rat somatostatin gene, was termed cyclic AMP response element or CRE (Montminy et al., 1986). The CRE octamer displays properties of a classic enhancer sequence, regulating transcription at a distance and functioning independent of orientation (Montminy et al., 1990). In 1993, evidence was presented that for maximal cAMP responsiveness, an additional conserved element, four to six nucleotides downstream of the CRE with the consensus C/GAGA/C, was needed (Kwast-Welfeld et al., 1993). PKA is a tetrameric enzyme possessing two catalytic and two regulatory subunits. In mammals, upon cAMP binding to the regulatory subunit, the catalytic subunit dissociates and migrates to the nucleus where it phosphorylates serine 133 and thereby activates CREB. Phosphorylation of this site does not seem to influence the DNA-binding aYnity of CREB, but is thought to activate the trans-activating domain by altering its conformation, thereby enhancing the interaction with the transcription machinery. This CREB activation is rapid, generally peaking within 30 min and declining gradually over 24 h (Montminy et al., 1990). Phosphorylated CREB was later shown to bind the nuclear protein CBP (CREB-binding protein) (Chrivia et al., 1993; Parker et al., 1996) and its paralog p300 (Eckner et al., 1994), which have intrinsic histone acetyltransferase activity (Ogryzko et al., 1996) and in addition provide bridging to the preinitiation complex (Chrivia et al., 1993; Kwok et al., 1994). Dephosphorylation of mammalian CREB is accomplished via nuclear phosphatases PP-1 (Alberts et al., 1994; Hagiwara et al., 1992) and PP2-A (Wadzinski et al., 1993). Whether and how phosphatase activity is controlled in a stimulus-inducible manner are matters that are still relatively poorly understood. It is well established that CREB has both constitutive and cAMP-inducible activities, with distinct domains within the protein contributing to these activities. The basal activity of CREB maps to a rather carboxy-terminal, glutamine-rich, constitutive activation domain (CAD or Q2), whereas phosphorylation and inducibility map to a central kinase-inducible domain (KID) (Fig. 2). The CAD domain interacts with and recruits the promoter recognition factor TFIID through association with a specific TATA box-binding protein-associated factor (Ferreri et al., 1994). CAD is both necessary and

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FIG. 2 Principal domains of the mammalian CREBa and the Drosophila CREB2-a protein. Q (1 and 2), glutamine-rich domains; a, a-domain; KID, kinase-inducible domain; CAD, constitutive activation domain; B, basic domain; Zip, leucine zipper domain. Numbers on top indicate the position of the amino acid residues. CREBa is one of the major products of the CREB gene.

FIG. 3 Transcription factors that are interacting with CREB. RNA polymerase II transcribes a gene (horizontal line, with the transcription start indicated by an arrow) that possesses a TATA box and an upstream cAMP response element (CRE). CREB binds the CRE as a dimer and interacts via its glutamine-rich region (Q2) with the basal transcription machinery of the cell, i.e., transcription factor IIB (TFIIB) and TATA box-binding protein (TBP)-associated factor 130 (TAF130). The phosphorylated kinase-inducible domain (KID) of CREB interacts with the KIX domain in CREB-binding protein (CBP), or potentially a CBP dimer. CBP associates indirectly with RNA polymerase II (Pol II) via the RNA helicase A protein (RHA). [Reprinted, with permission, from Shaywitz and Greenberg (1999). Annu. Rev. Biochem. 68, 821–861. #1999 by Annual Reviews.]

suYcient for basal transcription. The Ser-133-phosporylated KID recruits CBP. The CAD and KID domains act together in transcription activation whereby CAD recruits polymerase by interaction with TFIID and further activation of the polymerase is achieved by phosphorylated KID (Asahara et al., 2001; Felinski and Quinn, 2001; Shaywitz and Greenberg, 1999) (Fig. 3).

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B. Cyclic AMP Response Element Binding Proteins in Drosophila In 1992, two groups independently identified a CREB-like protein in Drosophila. Abel et al. described a transcription factor that binds to fat bodyspecific enhancers of alcohol dehydrogenase and yolk protein genes. They termed this 56-kDa protein box B-binding factor-2 (BBF-2), since it was isolated by screening a Drosophila embryonic gt11 expression library with a probe containing multiple copies of box B, the larval fat body enhancer of Drosophila mulleri (Abel et al., 1992). In the same year Smolik et al. cloned a CREB-like protein, which they designated dCREB-A, by using a somatostatin CRE oligo as a probe in a similar gt11 library screen. Both proteins were shown to belong to the family of bZip proteins, were able to bind to CREs, and activated transcription in a cellular system (Smolik et al., 1992). The two names are now used for the same protein. The basic region closely resembles that of mammalian CREB, while the leucine zipper domain is unusual because the third leucine is substituted by a tyrosine residue and it contains six instead of the four heptad repeats found in other CREB zippers. dCREB-A does not contain PKA phosphorylation sites and while it was shown to be a transcriptional activator in cell culture, it does not stimulate additional transcription in response to cAMP (Smolik et al., 1992). It does possess glycogen synthase kinase-3 phosphorylation sites, casein kinase I and II sites, and three calcium/calmodulin (CaM)-dependent kinase II sites and is a substrate for this kinase in vitro. A second member of the CREB family was identified in Drosophila by Usui et al. (1993) (dCREB-2). A cDNA with an open reading frame (ORF) coding for 285 amino acids was obtained, divided into four exons. It contained PKA, PKC, and CaM kinase II phosphorylation sites, but was unable to activate transcription of CRE-reported genes, either in the presence or absence of PKA. Later, it was shown that this exon arrangement was an alternative splice form of the dCREB2 gene (Yin et al., 1995b). The Drosophila CREB2 gene contains seven exons and seven splice variants termed dCREB2-a, -b, -c, -d, -q, -r, and -s. Only the dCREB2-a isoform, predicting a protein of 360 amino acids, is a PKA-dependent activator of transcription in cell culture. dCREB2-b (exons 1, 3, 5, and 7), on the other hand, is a transcriptional antagonist of PKA-dependent activation by dCREB2-a. The dCREB2-c (exons 1, 3, 4, 5, and 7) and -d isoforms (exons 1, 3, 4, 5, 6, and 7) are not activators but do contain a bZip domain. The short dCREB2-q, -r, and -s splice forms incorporate in-frame stop codons, resulting in predicted proteins that are truncated amino terminals to the bZip region. We recently identified an additional splice variant of the dCREB2 gene in the Drosophila Schneider 2 cell line (Schneider, 1972), possessing all exons except exons 2 and 4, that was termed dCREB2-e (Poels et al., 2004; Fig. 4). This isoform diVers from dCREB2-d by the absence of the very short

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FIG. 4 General exon arrangement of five splice variants of the dCREB2 gene that still contain the bZip domain (exon 7). Exon length in base pairs (bp) is indicated in parentheses. Intron lengths are indicated with triangles on top of the sequence. The novel dCREB2-e form was identified in Drosophila Schneider 2 cells. The kinase-inducible domain resides in exon 5 (Poels et al., 2004; Yin et al., 1995b). [Reprinted from Poels, J., Franssens, V., Van Loy, T., Martinez, A., Suner, M., Dunbar, S. J., De Loof, A., and Vanden Broeck, J. (2004). Isoforms of cyclic AMP response element binding proteins in Drosophila S2 cells. Biochem. Biophys. Res. Commun. 320, 318–324. Copyright (2004), with permission from Elsevier Science.]

exon 4 (four amino acids). It does not likely code for a PKA-responsive activator, since the activator form (dCREB2-a) requires both exons 2 and 6. In addition, the slightly longer dCREB2-d isoform is not active in cotransfection assays (Yin et al., 1995b). It does contain a bZip domain, glutaminerich stretches, and a PKA consensus phosphorylation site. Since the shorter dCREB2-b isoform works as a direct antagonist of PKA-dependent activation by dCREB2-a, this might also be the case for the dCREB2-e protein. This inhibition can either result from binding to and hence occupying the available CREs (or diminishing the available cofactors that are necessary for CREB-induced transcription) or from direct contact with dCREB2-a (dimerization). Alternative splicing also occurs in the mammalian CREB and CREM genes giving rise to many diVerent isoforms, either activators or repressors. The CREM gene isoforms are not expressed uniformly across tissues (Sassone-Corsi, 1995). This corresponds to the situation found in the Drosophila dCREB2 gene where Northern blots from diVerent fly tissues and stages show a complex pattern of bands with only two transcripts of 0.8 and 3.5 kb common to all stages. Recently, it became clear that regulation of dCREB2 activity diVers from mammalian CREB regulation. The KID domain of dCREB2 contains phosphorylation sites that are conserved in the corresponding domain of mammalian CREB. While in mammals phosphorylation of serine 133 (Ser-133) by PKA allows CREB to interact with CBP, it seems that in Drosophila most of the dCREB2 proteins are in a phosphorylated state at the corresponding

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PKA-site (Ser-231). Furthermore, it was found that the dCREB2 protein exists in a phosphorylated state that is unable to bind DNA and that this inhibition can be relieved by phosphatase treatment. The kinase responsible for this phosphorylation-dependent inhibition of DNA binding was shown to be casein kinase (CK) II, a ubiquitous serine/threonine kinase. PKA or calcium/calmodulin-dependent kinase II had no eVect on DNA binding. A model was proposed that predicts that inactive dCREB2 exists in flies with the PKA and CK sites phosphorylated. For dCREB2 to become activated, dephosphorylation of the CK sites (and probably of Ser-231, since sequence specificity for serine phosphatase has generally not been demonstrated) is required, which is followed by subsequent DNA binding and phosphorylation of Ser-231. Inactivation of dCREB2 would then be accomplished by phosphorylation of the CK sites (Horiuchi et al., 2004). A second specific CRE-binding complex was detected in Drosophila heads in this study. The DNA-binding properties of this complex were independent of phosphatase treatment. Whether this protein complex corresponds to dCREB-A was not specified.

C. Functions of Drosophila CREB Proteins The dCREB-A transcripts have a well-defined temporal and spatial pattern of expression. A developmental Northern analysis suggests that dCREB-A mRNA is present in embryos up to hour 16 of development and in very low levels in the three larval instars or pupae. In adults, it is detected at low levels in the brain, optic lobe, and midgut, while higher levels are seen in the salivary glands. One interesting feature of dCREB-A expression is that it has a sexually dimorphic pattern. In the female, dCREB-A RNAs are found in the ovarian follicle cells and in the male, transcription is seen in the reproductive tract (Rose et al., 1997; Smolik et al., 1992). These results suggested that regulation of dCREB-A is complex and provided evidence for a possible role in fertility and in the visual system in the adult. Since the dCREB-A protein does not contain a PKA site, it is unlikely to be part of the cAMP signal transduction pathway that is involved in learning and memory. However, transcription in the brain and optic lobes suggests that it is needed for other neural functions. A role of dCREB-A in embryogenesis was provided by the generation of null mutants. Animals that no longer express dCREB-A protein die late in embryogenesis before or at hatching, but have no obvious developmental defects in most of the organs in which it is expressed. This suggests that dCREB-A may aVect only the terminal diVerentiation steps in these organs. It also demonstrates a nonredundant function for this CRE-binding protein (Rose et al., 1997). Loss-of-function mutations of the gene also aVect the

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salivary gland and epidermis (Andrew, 1998; Andrew et al., 1997). While the eVect on salivary glands was mild, the epidermis was more aVected. dCREB-A mutant larvae have weak and lateralized cuticle. The most ventral denticles are replaced by denticles normally found in a ventrolateral position, and the most dorsal hairs are replaced by hairs normally found in dorsolateral position. The altered cuticle pattern is not due to a failure to diVerentiate denticles and hairs, but rather arises because wild-type dCREB-A is required for patterning of the epidermis along the dorsal-ventral axis. While dCREB-A is expressed in specific tissues, the dCREB2 gene seems to be transcribed ubiquitously in the embryo (Usui et al., 1993). However, changes of particular isoforms are occurring on developmental Northern blots, suggesting that each of the transcripts may have diVerent functions in embryogenesis (Yin et al., 1995b). The cAMP-pathway has long been implicated in learning and the formation of memory in flies. Screening for learning-defective mutant flies revealed several genes that are involved in the cAMP pathway, such as dunce (dnc) (Dudai et al., 1976), rutabaga (rut) (Livingstone et al., 1984), and PKA catalytic and regulatory subunits (Drain et al., 1991). Cloning of the dunce gene revealed it to be a cAMP phosphodiesterase (Chen et al., 1986), while rutabaga encodes a Ca2þ/calmodulin-responsive adenylyl cyclase (Levin et al., 1992). In the adult fly, the dCREB2 gene has also been proven to be involved in long-term memory formation. Memory after olfactory learning in Drosophila consists of two components, a cycloheximide-sensitive, longterm memory (LTM) and a cycloheximide-insensitive, anesthesia-resistant memory (ARM). ARM decays away within 4 days, while LTM shows no decay over a 7-day period and is dependent on protein synthesis. Formation of LTM in flies, but not ARM, is disrupted by induced expression of a dominant negative transgene of dCREB2 (Yin et al., 1994). Mutating two amino acids in the leucine zipper domain of this repressor isoform (dCREB2-b, see Section III.B and Fig. 4) was suYcient to prevent the dominant negative eVect on LTM. Thus, induction of LTM is not only protein synthesis dependent, but also dCREB2 dependent. In addition, it was shown that induced expression of the dCREB2-a activator isoform in transgenic flies enhances the formation of LTM. This eVect also depends on the phosphorylation of dCREB2-a, since enhanced LTM was not observed in flies carrying a mutant isoform with the putative PKA phosphorylation site disabled (Yin et al., 1995a). Thus, the ratio of activator and repressor dCREB2 isoforms determines LTM in flies. The role of CREB during learning and memory has a cellular correlate in the function of CREB during long-term synaptic plasticity (Davis et al., 1996). In the Drosophila neuromuscular junction (NMJ), the region where a motor neuron contacts its target muscle, a distinction between structural and functional changes in synaptic transmission has been elucidated. An

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indication that cAMP is involved in the process of long-term synaptic plasticity originated from studies with dunce mutants (that possess elevated cAMP levels) where structural and functional eVects on the neuromuscular junction were detected (Zhong and Wu, 1991; Zhong et al., 1992). Structural expansion of the NMJ is a result of downregulation of the neural cell adhesion molecule Fasciclin II (FasII) in response to an increase in activity and cAMP (Schuster et al., 1996). However, these FasII-mediated changes are not suYcient to change synaptic strength. For a persistent modification in synaptic function, an increase in the activation of dCREB2 is required in parallel (Davis et al., 1996). Elevation of dCREB2 activity in the nucleus initiates transcription of genes to occupy the expanded synaptic structure with more release machinery and, thus, increase in presynaptic transmitter release (Brunner and O’Kane, 1997; Davis et al., 1996). The Jun/Fos heterodimer, AP-1, can positively regulate both synaptic strength and synapse number, indicating that AP-1 acts upstream of CREB and functions at the top of the hierarchical cascade that regulates long-term plasticity (Sanyal et al., 2002). AP-1 regulation of CREB can be achieved through transcriptional activation, since the CREB promoter region contains consensus AP-1binding sites. Combined with previous reports that state that CREB regulates AP-1 expression (Greenberg et al., 1992; Sheng et al., 1990; Wang and Goldstein, 1994), a model can be postulated in which CREB induction of AP-1 is considered part of a positive feedback loop (Sanyal et al., 2002). dCREB2 also plays a role in circadian rhythms. This was shown by constructing transgenic Drosophila lines carrying a luciferase reporter gene driven by an enhancer element comprised of CREB-binding sites. Luciferase activity measurements of these flies reported a 24 h oscillation both in a light– dark cycle and in constant darkness. The expression levels and cycling pattern of this reporter construct were dramatically reduced in flies that possessed a mutation in the dCREB2 gene. However, the dCREB2 protein itself did not appear to cycle in a circadian rhythm, nor was the phosphorylation state of Ser-231 (the PKA phosphorylation site) altered in a similar oscillation (Belvin et al., 1999). dCREB2 may have an unknown binding partner that cycles or a kinase that phosphorylates the protein at a residue other than Ser-231, and which aVects its activity, may cycle. These experiments showed that dCREB2 activity is under circadian control. In addition, normal circadian fluctuations in activity disappeared in the dCREB2 mutants, a phenotype that could be rescued by inducing dCREB2 expression. A direct eVect of the mutant dCREB2 gene was detected on the period protein (Per), the product of one of the clock genes (per) found in Drosophila, where the Per protein is present at more equal levels throughout the circadian cycle (instead of the wild-type cyclic expression pattern). The per promoter contains three CREs in the region required for normal Per expression. It is possible that dCREB2 contributes to Per expression through these sites. In

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conclusion, dCREB2 and Period aVect each other, suggesting that they are part of the same regulatory feedback loop (Belvin et al., 1999). Normal clockcontrolled CREB activity is also influenced by dFMR1, an RNA-binding protein. Adult dfmr1 mutant flies display arrhythmic circadian activity that is not linked to a defect in the expression of the clock components. In these dfmr1 mutant flies, cycling of a CRE-luciferase reporter is detected, but with a clearly reduced amplitude of the oscillations. In addition, dfmr1 mutant males display reduced courtship activity (DockendorV et al., 2002). A molecular link between memory and circadian rhythms can also be fulfilled by dCREB2 (Waddell and Quinn, 2001). In mammals, sleep is important for memory consolidation. Flies also possess prolonged periods of inactivity (Hendricks et al., 2000). Since dCREB2 activity cycles with a peak occurring just after darkness, it is possible that rest drives CREB activity levels and thereby promotes memory consolidation (Waddell and Quinn, 2001). Results supporting a functional role for CREB in a restorative function of the sleep-like rest state of Drosophila during recovery from rest deprivation were obtained by Hendricks et al. (2001). In the embryo, a CRE was identified within the ultrabithorax (Ubx) midgut enhancer as a target sequence for decapentaplegic (Dpp) signaling. In addition, multiple CREs drive endoderm expression from a labial enhancer, a homeotic gene that is also transcriptionally regulated by Dpp. Dpp is a Drosophila growth factor of the TGF-b family with multiple functions during embryonic development, such as endoderm induction where Dpp plays a key role. Evidence was further presented for a CREB protein to be a target transcription factor for Dpp signaling in the embryonic midgut (Eresh et al., 1997). The dCREB2 isoforms, rather than dCREB-A, are good candidates for transcription factor targets, based on their uniform expression in the embryonic midgut, while dCREB-A expression seems to be absent in this tissue. Finally, a role for the dCREB2-b isoform in female sexual receptivity was recently presented by Sakai and Kidokoro (2002). Wild-type males in this study mated more quickly with females in which dCREB2-b was overexpressed than with control females. Overexpression of dCREB2-a did not change mating frequency or courtship behavior.

D. bZip and CREB Proteins in Other Invertebrate Species No bZip proteins, except for CREB-related proteins, have been cloned from other insect species. This fact displays a current gap in our knowledge about the evolution, regulation, and role of this transcription factor family in this vast species group. Questions remain concerning diVerent roles played by, e.g., some of the bZip genes involved in larval development of Drosophila and

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their function in heterometabolous insects (insects undergoing incomplete metamorphosis in which the nymph is essentially like the adult and there is no pupal stage) such as locusts. In the malaria mosquito, Anopheles gambiae, at least 25 putative bZip-like proteins can be found. Most of these possess orthologues in Drosophila melanogaster, but no functional data are available for any of these genes. Figure 5 shows a neighbor joining tree of predicted Drosophila and Anopheles bZip proteins. So far, only two CREB genes have been cloned from another insect, i.e., Apis mellifera (Eisenhardt et al., 2003) and Aedes aegypti (Dittmer et al., 2003). Similar to the Drosophila dCREB2 gene, the AmCREB gene encodes at least eight splicing variants (Fig. 6). It contains two glutamine-rich regions, thought to represent the functional activator domains of CREB proteins, and a kinase inducible domain that possesses PKA, PKC, and CK II phosphorylation sites. Immunohistochemical analyses located AmCREB isoforms in brain structures that are likely involved in memory processes in the honeybee, indicating a function of CREB in the formation of memory components that depend on transcription and translation (Eisenhardt et al., 2003; Gru¨ nbaum and Mu¨ ller, 1998; Wu¨ stenberg et al., 1998). Table II displays diVerent invertebrate CREBs and their role in the respective organism, as discussed in the text. A classic example of CREB being involved in learning can be found in the sea slug Aplysia californica, a marine invertebrate with a relatively simple nervous system (Fig. 7). This animal responds to a tactile stimulus by withdrawing its gill and siphon, organs normally exposed to the environment. This withdrawal response to a tactile stimulus is faster and more robust if the animal first receives a noxious-sensitizing stimulus, such as a shock to the tail (Mayford and Kandel, 1999). The tactile stimulus is sensed by somatosensory neurons that connect directly to motor neurons that control the muscles involved in the withdrawal response. The synapse between the involved sensory and motor neurons can be reconstituted in primary cell culture. The noxious stimulus activates neurons that release serotonin and synapse onto the sensory neurons, where serotonin binds its corresponding G protein-coupled receptor and thereby activates adenylyl cyclase, leading to an increase in cAMP. The synapse between the sensory and the motor neuron undergoes short-term facilitation in response to one pulse of serotonin, whereas long-term facilitation is achieved after five pulses of serotonin. This last process requires transcription and translation and can be inhibited by injecting CRE oligonucleotides into the nucleus of sensory neurons (Alberini et al., 1994; Dash et al., 1990). Aplysia sensory neurons constitutively express ApCREB2, a leucine zipper transcription factor that is partially homologous to human CREB2 (Bartsch et al., 1995). ApCREB2 acts as a repressor for the morphological as well as the functional changes that accompany long-term facilitation. The Aplysia

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FIG. 5 Neighbor-joining tree showing putative orthologues of Drosophila melanogaster (gi-number from GenBank beginning with 2 or 7) and Anopheles gambiae (ENSANGP number) bZip proteins. This phylogram was produced from a multiple alignment of the bZip region of the proteins. Redundant sequences were excluded from the alignment. Sisterless-A and Apontic were also not included because of their unusual (putative) bZip domains. Bootstrap support (1000 replicas) is indicated in percentage.

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FIG. 6 Amino acid sequence comparison of the bZip domains of some insect CREB proteins, i.e., Drosophila melanogaster (DmCREBA and DmCREB2), the malaria mosquito Anopheles gambiae (AgCREB1 ¼ ENSANGP00000003100 and AgCREB2 ¼ ENSANGP00000026797), the yellow fever mosquito Aedes aegypti (AaCREB), and the honeybee Apis mellifera (AmCREB) CREB-like proteins. Boxed residues represent the conserved leucines in the leucine zipper domains, except for AgCREB1 and DmCREBA where a tyrosine replaces the third leucine residue.

TABLE II The CREB-Related Genes of Several Invertebrate Speciesa Leucine zipper structure

Biological functions/ involved processes

dCREB-A

LxLxYxLxLxL

Salivary gland and epidermis development

dCREB2

LxLxLxL

Learning and memory, long-term synaptic plasticity at the NMJ, circadian rhythm, rest homeostasis, midgut development, female sexual receptivity

AmCREB

LxLxLxL

Putatively involved in learning and memory

ApCREB1

LxLxLxL

Long-term facilitation

Gene

LymCREB1

LxLxLxL

LymCREB2

IxLxLxL

HydCREB

LxLxLxL

Long-term synaptic plasticity Regeneration

a CREB genes isolated from several invertebrate species (column 1) and the leucine zipper structure given in single character amino acid code, where ‘‘x’’ represents a stretch of six amino acids (column 2). The third column displays possible biological functions for the corresponding CREB isoforms, as discussed in the text.

CREB1 gene encodes a nuclear activator (CREB1a) that interacts with ApCREB2 (Choi et al., 2003), and a nuclear repressor (CREB1b). Blocking the expression of CREB1a impairs long-term facilitation, while after blocking either ApCREB2 or CREB1b, one single pulse of serotonin can induce long-term facilitation lasting one or more days (Bartsch et al., 1998). Downstream targets of Aplysia CREB are immediate response genes, including ubiquitin hydrolase and the bZip protein C/EBP (Alberini et al., 1994; Hegde et al., 1997). Ubiquitin hydrolase plays a role in the degradation of the regulatory subunit of PKA, leading to a persistent increase in the activity

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FIG. 7 Molecular signaling for long-term synaptic facilitation in Aplysia, during sensitization of the gill-withdrawal reflex. See text for details. [Reprinted, with permission, from Abel, T., Martin, K.C., Bartsch, D., and Kandel, E.R. (1998). Science 279, 338–341. Copyright 1998 AAAS.]

of the catalytic subunits (Hegde et al., 1993). ApC/EBP mediates growth of new synaptic connections (Alberini et al., 1994). The genetic switch from short-term to long-term facilitation can be divided into three components: (1) the removal of ApCREB2-mediated repression and activation of CREB1, (2) induction of immediate response genes, and (3) growth of new connections (Abel et al., 1998). ApC/EBP can be degraded by the ubiquitin–proteasome pathway, whereby phosphorylation of C/EBP by MAP kinase protects the transcription factor from proteolysis (Yamamoto et al., 1999). ApC/EBP can also interact with Aplysia activating factor

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(ApAF), another bZip protein that can heterodimerize with ApCREB2 as well, but not with CREB1. ApAF is a transcriptional activator that is constitutively expressed in Aplysia sensory neurons (unlike ApC/EBP that is induced upon stimulation with serotonin) and can be modulated by PKA. ApAF is critical for enhancing and stabilizing gene expression changes induced by phosphorylated CREB1a and derepression of ApCREB2 (Bartsch et al., 2000). Interestingly, Aplysia cell adhesion molecules (apCAMs) are downregulated upon repeated exposure to serotonin (Bailey et al., 1992, 1997). ApCAMs belong to the same protein family as Drosophila Fasciclin II (see also Section III.C), an adhesion molecule that is downregulated in the Drosophila NMJ in response to an increase in activity and cAMP. The selective down-regulation of the transmembrane isoform of apCAM decreases the interaction of sensory cell neurites with each other and leads to the formation of new synaptic connections (Abel et al., 1998; Zhu et al., 1994, 1995). Whereas multiple serotonin pulses produce long-term facilitation, repeated pulses of the neuropeptide FMRFa produce long-term depression of the synapse between the sensory and motor neurons in Aplysia (Montarolo et al., 1988). Thus, long-term synaptic plasticity can be regulated in two directions. The mechanisms responsible for this regulation have been elucidated by studying the chromatin structure around the promoter of ApC/EBP. As already indicated, serotonin induces ApC/EBP expression by activating CREB1a through PKA phosphorylation, which recruits CREBbinding protein (CBP, see also Section III.A). CBP then acetylates histones around the ApC/EBP promoter. Acetylation of histone tails is believed to facilitate the unraveling of chromatin and in turn increases the accessibility of the local chromatin to transcriptional machinery complexes (Lonze and Ginty, 2002). In contrast, the inhibitory neurotransmitter FMRFa displaces CREB1a from the promoter by recruiting ApCREB2. This leads to recruitment of the histone deacetylase HDAC-5 that alters the chromatin structure and represses C/EBP, probably by making the chromatin structure more dense. When the two opposite stimuli are applied together, the eVect of FMRFa dominates and CREB1a is displaced from the promoter, leading to ApC/EBP repression (Guan et al., 2002). CREB protein homologues are also present in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is an established model system for studying cellular mechanisms of behavioral learning at the level of individually identifiable neurons (Ribeiro et al., 2003). Lymnaea CREB1 is the homologue of the transcriptional activator Aplysia CREB1a, while Lymnaea CREB2 is homologous to the transcriptional repressor ApCREB2. Also in this organism, injecting a CRE oligonucleotide in a neuron that contains Lymnaea CREB1 mRNA inhibited cAMP-induced, long-term synaptic plasticity (Sadamoto et al., 2004).

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Another function for a CREB protein was found in hydra (Galliot et al., 1995). Hydra, which belong to the phylum of Cnidaria, display regenerative capacities. During regeneration, modifications in gene expression have been identified (Galliot, 2000). Recently, it was found that the number of phospho-CREB-expressing cells in head regenerating tips was dramatically increased during hydra regeneration (Kaloulis et al., 2004). A putative hydra CREB regulator thereby is the p80 kinase that belongs to the ribosomal protein S6 kinase family. Members of this family have been previously shown to phosphorylate CREB proteins (De Cesare et al., 1998; Pende et al., 1997; Pierrat et al., 1998; Xing et al., 1996).

IV. Concluding Remarks A vast amount of evidence correlates insect CREBs, as well as other invertebrate CREBs, with physiological processes that are very similar to the ones regulated by vertebrate CREB proteins. Invertebrates also produce several isoforms of their CREB proteins, either being activators or repressors of transcription. The CREBs found in invertebrates are also being regulated by phosphorylation via diVerent protein kinases, and by dephosphorylation via phosphoprotein phosphatases, in response to a large variety of extracellular stimuli. Despite many similarities, important diVerences in the regulation of CREB activation exist between these distantly related metazoan phyla, as was recently demonstrated (Horiuchi et al., 2004). While phosphorylation of CREB proteins has been specified in a massive amount of studies, much less is currently known about the necessary steps and the regulation of the dephosphorylation of CREBs. While Drosophila CREB proteins, and Drosophila bZip proteins in general, have been studied extensively, little is known about corresponding bZip proteins in other insect species. Questions remain as to whether orthologues for these Drosophila proteins exist in other insects, e.g., as seems to be the case for the malaria mosquito Anopheles gambiae. Whether these orthologues possess similar biological functions is still largely unknown. It seems not unlikely that insects that belong to diVerent subgroups (e.g., holometabolous versus heterometabolous insects) have evolved specific roles for some of their bZip transcription factors. Even within one insect species, bZip proteins can function in one tissue, while being largely inactive in another. The reasons for this discrepancy probably lie in diVerences of the bZip spectrum that is being produced by a certain tissue. Combinations of diVerent bZip proteins may thereby acquire a diVerent DNA recognition motif. Also, cell- or tissue-specific basal transcription factors play a role in bZip-mediated gene transcription.

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One common theme found in all invertebrate CREB genes being cloned so far is that one or more of the CREB isoforms seems to be involved in memory and synaptic facilitation. The focus on bZip-regulated transcription is now shifting toward downstream factors that are being up- or downregulated in response to diVerent stimuli. Undoubtedly, m-array analysis will be of great importance in resolving the time- and space-related diVerential gene transcription profiles in processes where bZip proteins are involved. In conclusion, insects possess a remarkably complex array of bZip transcription factors. For many of these factors, the biological relevance is not known. Drosophila melanogaster, with its highly evolved genetic tools, will certainly aid in solving questions that apply to the regulation and downstream gene transcription eVects of bZip proteins. Studies on Drosophila and other insects will thereby provide indications for putative roles in mammalian organisms. After all, it seems that insects, with their tiny nervous systems, are capable of learning and memorizing, tasks that were once considered to be specifically human.

Acknowledgments The authors gratefully acknowledge the Belgian ‘‘Interuniversity Attraction Poles Programme’’ (IUAP/PAI P5/30, Belgian Science Policy) and the ‘‘FWO-Vlaanderen’’ for financial support.

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