NORMAL GENOMIC

• Fontenele M et al.
• Calpain A modulates Toll responses by limited Cactus/IkappaB proteolysis.
• Mol Biol Cell. 2013; 24: 2966-80
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• Calcium-dependent cysteine proteases of the calpain family are modulatory proteases that cleave their substrates in a limited manner. Among their substrates, calpains target vertebrate and invertebrate IkappaB proteins. Because proteolysis by calpains potentially generates novel protein functions, it is important to understand how this affects NFkappaB activity. We investigate the action of Calpain A (CalpA) on the Drosophila melanogaster IkappaB homologue Cactus in vivo. CalpA alters the absolute amounts of Cactus protein. Our data indicate, however, that CalpA uses additional mechanisms to regulate NFkappaB function. We provide evidence that CalpA interacts physically with Cactus, recognizing a Cactus pool that is not bound to Dorsal, a fly NFkappaB/Rel homologue. We show that proteolytic cleavage by CalpA generates Cactus fragments lacking an N-terminal region required for Toll responsiveness. These fragments are generated in vivo and display properties distinct from those of full-length Cactus. We propose that CalpA targets free Cactus, which is incorporated into and modulates Toll-responsive complexes in the embryo and immune system.

• Li C et al.
• Identification, characterization, and function analysis of the Cactus gene from Litopenaeus vannamei.
• PLoS One. 2012; 7: 49711-49711
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• The nuclear factor-kappa B (NF-kappaB) pathways play important roles in innate immune responses. IkappaB is the main cytoplasmic inhibitor of NF-kappaB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IkappaB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-kappaB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-kappaB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV) infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.

• Hughes AL
• Evolution of the betaGRP/GNBP/beta-1,3-glucanase family of insects.
• Immunogenetics. 2012; 64: 549-58
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• The betaGRP/GNBP/beta-1,3-glucanase protein family of insects includes several proteins involved in innate immune recognition, such as the beta-glucan recognition proteins of Lepidoptera and the Gram-negative bacteria-binding proteins of Drosophila. A phylogenetic analysis supported the existence of two distinct subfamilies, designated the pattern recognition receptor (PRR) and glucanase subfamilies, which originated by gene duplication prior to the origin of the Holometabola. In the C-terminal region (CTR) shared by both subfamilies, the PRR subfamily has evolved significantly more rapidly at the amino acid sequence level than has the glucanase subfamily, implying a relative lack of constraint on the amino acid sequence of this region in the PRR subfamily. PRR subfamily members also include an N-terminal region (NTR), involved in carbohydrate recognition, which is not shared by glucanase subfamily members. In comparisons between paralogous PRR subfamily members, there were no conserved amino acid residues in the NTR. However, when pairs of putatively orthologous PRR subfamily members were compared, the NTR was most often as conserved as the CTR or more so. This pattern suggests that the NTR may be important in functions specific to the different paralogs, while amino acid sequence changes in the NTR may have been important in functional differentiation among paralogs, specifically with regard to the types of carbohydrates that they recognize.

• Tannoury H, Rodriguez V, Kovacevic I, Ibourk M, Lee M, Cram EJ
• CACN-1/Cactin interacts genetically with MIG-2 GTPase signaling to control distal tip cell migration in C. elegans.
• Dev Biol. 2010; 341: 176-85
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• The two specialized C. elegans distal tip cells (DTCs) provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC migration. RNAi depletion experiments and analysis of the hypomorphic allele cacn-1(tm3126) indicate that CACN-1 is required during DTC migration for proper pathfinding and for cessation of DTC migration at the end of larval morphogenesis. Strong expression of CACN-1 in the DTCs, and data from cell-specific RNAi depletion experiments, suggest that CACN-1 is required cell-autonomously to control DTC migration. Importantly, genetic interaction data with Rac GTPase activators and effectors suggest that CACN-1 acts specifically to inhibit the mig-2/Rac pathway, and in parallel to ced-10/Rac, to control DTC pathfinding.

• Atzei P, Yang F, Collery R, Kennedy BN, Moynagh PN
• Characterisation of expression patterns and functional role of Cactin in early zebrafish development.
• Gene Expr Patterns. 2010; 10: 199-206
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• The immune system of teleost zebrafish (Danio rerio) shows high similarity to mammalian counterparts sharing many innate immune components including Toll-Like Receptors (TLRs), cytokines, chemokines and complement molecules. As in mammals, zebrafish also contains the transcription factor NF-kappaB that plays dualist roles in innate immunity and early development. Indeed NF-kappaB members are expressed in different temporal patterns during the early stages of zebrafish embryogenesis indicating that each molecule is involved in specific developmental events. In the present study we employ zebrafish as a model to characterise the expression pattern and role of a novel NF-kappaB regulator, termed Cactin, in early development. Cactin was first characterised in Drosophila as a new member of the Rel pathway that could affect the generation of dorsal-ventral polarity. To explore the potential developmental role of Cactin in zebrafish, we initially investigated its expression pattern and functional role during early embryonic developmental stages. We detect Cactin expression at all stages of early development and knockdown of Cactin by specific morpholino antisense oligonucleotides causes developmental abnormalities manifested by an overall dysmorphic cellular organisation. These results indicate that Cactin has been highly conserved during evolution and plays a key role in early embryonic development.

• Range RC, Venuti JM, McClay DR
• LvGroucho and nuclear beta-catenin functionally compete for Tcf binding to influence activation of the endomesoderm gene regulatory network in the sea urchin embryo.
• Dev Biol. 2005; 279: 252-67
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• In the sea urchin embryo, specification of the endomesoderm is accomplished by the activity of a network of regulatory genes in the vegetal hemisphere, called the endomesoderm gene regulatory network (GRN). The activation of this network is mediated primarily through the activity of the Wnt pathway, though details of pathway activation remain unclear. To gain further insight into control of endomesoderm GRN activation, we have identified a sea urchin homologue of the co-repressor Groucho (LvGroucho) that has been shown to antagonize beta-catenin/Tcf activation complexes during Wnt signaling in other systems. Groucho functions by recruiting the histone deacetylase Rpd3 to the DNA template via interaction with site-specific transcription factors, resulting in localized chromatin condensation and transcriptional silencing. Our results show that the LvGroucho protein localizes to all nuclei throughout embryonic development. Interaction assays demonstrate that LvGroucho interacts with Tcf via both the Q and the WD domains of the protein. LvGroucho interacts with Tcf to antagonize the expression of key endomesoderm regulatory genes. Assays demonstrate that LvGroucho and n beta-catenin functionally compete for binding to Tcf as a major mechanism by which the Tcf-control switch is regulated. Functional analysis of the N-terminal AES197 domain of LvGroucho shows that it is sufficient to recapitulate the function of full-length LvGroucho. This finding strongly supports the conclusion that the effects of LvGro overexpression are due primarily to its interactions with Tcf and not other Groucho interacting partners, since Tcf is the only protein present in the sea urchin known to interact with AES197. Because the Q domain is unable to bind Rpd3, it was expected to behave as a dominant negative LvGroucho. Unexpectedly, overexpression of the Q domain gave functional results similar to LvGroucho and the AES197 domain. This is the first evidence for an inherent repressive function for the Q domain alone. Together, our results indicate that LvGroucho functionally competes with beta-catenin for Tcf binding, and this competitive mechanism regulates one of the earliest steps in the initiation of the sea urchin endomesoderm GRN.

• Handel K, Basal A, Fan X, Roth S
• Tribolium castaneum twist: gastrulation and mesoderm formation in a short-germ beetle.
• Dev Genes Evol. 2005; 215: 13-31
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• Mesoderm formation has been extensively analyzed in the long-germ insect Drosophila melanogaster. In Drosophila, both the invagination and specification of the mesoderm is controlled by twist. Here we present a detailed description of mesoderm formation and twist regulation for the short-germ beetle Tribolium castaneum. In contrast to Drosophila, (1) the presumptive mesodermal cells of Tribolium are part of a mitotic domain and divide prior to ventral furrow formation, (2) ventral furrow formation progresses from posterior to anterior, (3) the number of cell layers within the furrow changes from multilayered in caudal to single layered in cephalic regions, and (4) there is a continuous production of mesodermal cells after gastrulation as new segments arise from the posterior growth zone. Tribolium twist (Tc-twist) is initially expressed in all presumptive mesodermal cells; however, after invagination, expression is maintained only in particular locations, which include the anterior compartments of the cephalic segments and a patch of cells at the posterior rim of the growth zone. The growth zone is multilayered with its inner cell layer being continuous with the mesoderm of the newly forming segments where twist expression is re-initiated anterior to the emerging engrailed stripes. A genomic region of Tc-twist was identified which drives ventral expression of a reporter construct in Drosophila. The expression of this Tc-twist construct in the background of Drosophila maternal mutations suggests that the dorsoventral system regulates Tc-twist, but that differences exist in regulation of the Dm-twist and Tc-twist genes by the terminal system.

• Novy M et al.
• EAPP, a novel E2F binding protein that modulates E2F-dependent transcription.
• Mol Biol Cell. 2005; 16: 2181-90
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• E2F transcription factors play an essential role in cell proliferation and apoptosis and their activity is frequently deregulated in human cancers. In a yeast two-hybrid screen we identified a novel E2F-binding protein. Due to its strong phosphorylation we named it EAPP (e2F-associated phosphoprotein). EAPP is localized in the nucleus and interacts with E2F-1, E2F-2, and E2F-3, but not with E2F-4. Examination of a number of human cell lines revealed that EAPP levels are elevated in most transformed cells. Moreover, EAPP mRNA was detected in all investigated human tissues in varying amounts. EAPP is present throughout the cell cycle but disappears during mitosis. In transfection assays with reporters controlled by either an artificial E2F-dependent promoter or the murine thymidine kinase promoter, EAPP increased the activation caused by E2F-1 but not by E2F-4. Surprisingly, the promoter of the p14(ARF) gene, which was also activated by E2F-1, became repressed by EAPP. Overexpression of EAPP in U2OS cells resulted in a significant increase of cells in S-phase, whereas RNAi-mediated knock down of EAPP reduced the fraction of cells in S-phase. Taken together, these data suggest that EAPP modulates E2F-regulated transcription, stimulates proliferation, and may be involved in the malignant transformation of cells.

• Yu F, Morin X, Kaushik R, Bahri S, Yang X, Chia W
• A mouse homologue of Drosophila pins can asymmetrically localize and substitute for pins function in Drosophila neuroblasts.
• J Cell Sci. 2003; 116: 887-96
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• Asymmetric cell division is a fundamental mechanism used to generate cellular diversity in invertebrates and vertebrates. In Drosophila, asymmetric division of neuroblasts is achieved by the asymmetric segregation of cell fate determinants Prospero and Numb into the basal daughter cell. Asymmetric segregation of cell fate determinants requires an apically localized protein complex that includes Inscuteable, Pins, Bazooka, DmPar-6, DaPKC and Galphai. Pins acts to stabilize the apical complex during neuroblast divisions. Pins interacts and colocalizes with Inscuteable, as well as maintaining its apical localization. We have isolated a mouse homologue of pins (Pins) and characterized its expression profile. Mouse PINS shares high similarity in sequence and structure with Pins and other Pins-like proteins from mammals. Pins is expressed in many mouse tissues but its expression is enriched in the ventricular zone of the developing central nervous systems. PINS localizes asymmetrically to the apical cortex of mitotic neuroblasts when ectopically expressed in Drosophila embryos. Like Pins, its N-terminal tetratricopeptide repeats can directly interact with the asymmetric localization domain of Insc, and its C-terminal GoLoco-containing region can direct localization to the neuroblast cortex. We further show that Pins can fulfill all aspects of pins function in Drosophila neuroblast asymmetric cell divisions. Our results suggest a conservation of function between the fly and mammalian Pins homologues.

• Bolatto C, Chifflet S, Megighian A, Cantera R
• Synaptic activity modifies the levels of Dorsal and Cactus at the neuromuscular junction of Drosophila.
• J Neurobiol. 2003; 54: 525-36
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• The Drosophila Rel transcription factor Dorsal and its inhibitor Cactus participate in a signal transduction pathway involved in several biologic processes, including embryonic pattern formation, immunity, and muscle development. In contrast with embryonic muscle, where Dorsal is reportedly absent, this protein and Cactus accumulates in the neuromuscular junctions in the muscle of both larvae and adults. The phenotype of homozygous dorsal mutant larvae suggested that Dorsal and Cactus maybe necessary for normal function and maintenance of the neuromuscular system. Here we investigate if these proteins can respond to synaptic activity. Using larval body wall preparations and antibodies specific for Dorsal or Cactus we show that the amount of these proteins at the neuromuscular junction is substantially decreased after electrical stimulation of the nerves or incubation in glutamate, the principal transmitter in this type of synapse. The specificity of the response was tested with a glutamate receptor antagonist (argiotoxin 636). Because the effect can be reproduced using a calcium ionophore (ionomycin treatment) as well as blocked by the inhibition of the muscle ryanodine receptor (tetracaine treatment), the involvement of calcium in this process seems likely. We also observed that the inhibition of the calcium dependent protein phosphatase calcineurin prevents the effect of glutamate on the fluorescence for Dorsal and Cactus, suggesting its participation in a signal transduction cascade that may activate Dorsal in the muscle independently of Toll. Our results are consistent with a novel function of the Rel factor Dorsal in a molecular pathway turned on by neural activity and/or contractile activity.

• Minakhina S, Yang J, Steward R
• Tamo selectively modulates nuclear import in Drosophila.
• Genes Cells. 2003; 8: 299-310
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• BACKGROUND: The NF-kappaB/Rel pathway functions in the establishment of dorsal-ventral polarity and in the innate humoral and cellular immune response in Drosophila. An important aspect of all NF-kappaB/Rel pathways is the translocation of the Rel proteins from the cytoplasm to the nucleus, where they function as transcription factors. RESULTS: We have identified a new protein, Tamo, which binds to Drosophila Rel protein Dorsal, but not to Dorsal lacking the nuclear localization sequence. Tamo does not bind to the other Drosophila Rel proteins, Dif and Relish. The Tamo-Dorsal complex forms in the cytoplasm and Tamo also interacts with a cytoplasmically orientated nucleoporin. In addition Tamo binds the Ras family small GTPase, Ran. Tamo functions during oogenesis and, based on phenotypic analysis, controls the levels of nuclear Dorsal in early embryos. It further regulates the accumulation of Dorsal in the nucleus after immune challenge. CONCLUSIONS: Tamo has an essential function during oogenesis. Tamo interacts with Dorsal and proteins that are part of the nuclear import machinery. We propose that tamo modulates the levels of import of Dorsal and other proteins.

• Heanue TA et al.
• Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.
• Mech Dev. 2002; 111: 75-87
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• We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.

• Kolsch V, Paululat A
• The highly conserved cardiogenic bHLH factor Hand is specifically expressed in circular visceral muscle progenitor cells and in all cell types of the dorsal vessel during Drosophila embryogenesis.
• Dev Genes Evol. 2002; 212: 473-85
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• The highly conserved basic helix-loop-helix transcription factor Hand plays a crucial role in cardiogenesis, limb formation and other developmental processes of vertebrates. Humans, mice and other higher vertebrates have two related genes, dHand (also known as Hand2, Hed, Thing2) and eHand (also known as Hand1, Hxt, Thing1), whereas fish and Drosophila have only a single hand gene. We cloned Drosophila hand and studied the embryonic expression in detail by using various tissue-specific markers that allowed us to analyze the identity of hand-expressing cells. We found hand to be expressed in the entire heart, including all cardioblasts and pericardial cells, in the progenitors of the circular visceral muscles, the lymph gland and garland cells, and in a few cells in the CNS. The expression of Drosophila hand starts after the inductive activity of the early regulators in these tissues, e.g. Tinman and Bagpipe, suggesting a role of Hand in differentiation rather than in tissue determination. In many aspects the expression pattern of Drosophila hand resembles the patterns of its vertebrates orthologues, for instance in cardiac tissues. We assume that Hand proteins might play a highly conserved role throughout evolution.

• Wakabayashi-Ito N, Belvin MP, Bluestein DA, Anderson KV
• fusilli, an essential gene with a maternal role in Drosophila embryonic dorsal-ventral patterning.
• Dev Biol. 2001; 229: 44-54
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• The Drosophila fusilli (fus) gene was identified in a genetic screen for dominant maternal enhancers of an unusual dorsalizing mutation in the cactus gene, cact(E10). While females that are heterozygous for the cact(E10) allele produce embryos with wild-type dorsal-ventral patterning, more than 90% of the embryos produced by females that are heterozygous for both cact(E10) and the fus(1) mutation are weakly dorsalized. Loss of fusilli activity causes lethality during embryogenesis but not dorsal-ventral patterning defects, indicating that fusilli is important in more than one developmental process. The fusilli gene encodes a protein with RNA binding motifs related to those in mammalian hnRNP F and H, which play roles in regulated RNA splicing. The fusilli RNA is not present in the oocyte or early embryo, and germ-line clones of fusilli mutations have no maternal effect on dorsal-ventral patterning, indicating that the fusilli maternal effect does not depend on germ-line expression of the gene. Because the fusilli RNA is present in ovarian follicle cells, we propose that fusilli acts downstream of the Drosophila EGF receptor to control the biogenesis of follicle cell transcripts that control the initial dorsal-ventral asymmetry of the embryo.

• Sonoda J, Wharton RP
• Drosophila Brain Tumor is a translational repressor.
• Genes Dev. 2001; 15: 762-73
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• The Drosophila brain tumor (brat) gene encodes a member of the conserved NHL family of proteins, which appear to regulate differentiation and growth in a variety of organisms. One of the founding family members, Caenorhabditis elegans LIN-41, is thought to control posttranscriptional gene expression. However, the mechanism by which LIN-41, or any other NHL protein, acts has not been clear. Using a yeast "four-hybrid" interaction assay, we show that Brain Tumor is recruited to hunchback (hb) mRNA through interactions with Nanos and Pumilio, which bind to the RNA to repress its translation. Interaction with the Nanos/Pumilio/RNA complex is mediated by the Brat NHL domain; single amino acid substitutions in this domain compromise quaternary complex assembly in vitro and hb regulation in vivo. Thus, recruitment of Brat is necessary for translational repression and the normal development of posterior embryonic pattern. In addition to regulating abdominal segmentation, previous genetic analysis has shown that Brat, Nanos, and Pumilio govern a variety of developmental processes. We examined the role of Brat in two of these processes-regulation of maternal Cyclin B mRNA in the embryo and regulation of imaginal disc development. The results of these experiments suggest that NHL domain proteins are recruited to various mRNAs by combinatorial protein-protein interactions.

• Boucher L, Ouzounis CA, Enright AJ, Blencowe BJ
• A genome-wide survey of RS domain proteins.
• RNA. 2001; 7: 1693-701
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• Domains rich in alternating arginine and serine residues (RS domains) are frequently found in metazoan proteins involved in pre-mRNA splicing. The RS domains of splicing factors associate with each other and are important for the formation of protein-protein interactions required for both constitutive and regulated splicing. The prevalence of the RS domain in splicing factors suggests that it might serve as a useful signature for the identification of new proteins that function in pre-mRNA processing, although it remains to be determined whether RS domains also participate in other cellular functions. Using database search and sequence clustering methods, we have identified and categorized RS domain proteins encoded within the entire genomes of Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae. This genome-wide survey revealed a surprising complexity of RS domain proteins in metazoans with functions associated with chromatin structure, transcription by RNA polymerase II, cell cycle, and cell structure, as well as pre-mRNA processing. Also identified were RS domain proteins in S. cerevisiae with functions associated with cell structure, osmotic regulation, and cell cycle progression. The results thus demonstrate an effective strategy for the genomic mining of RS domain proteins. The identification of many new proteins using this strategy has provided a database of factors that are candidates for forming RS domain-mediated interactions associated with different steps in pre-mRNA processing, in addition to other cellular functions.

• Patel NH, Hayward DC, Lall S, Pirkl NR, DiPietro D, Ball EE
• Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation.
• Development. 2001; 128: 3459-72
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• While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.

• Goel M, Garcia R, Estacion M, Schilling WP
• Regulation of Drosophila TRPL channels by immunophilin FKBP59.
• J Biol Chem. 2001; 276: 38762-73
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• Transient receptor potential and transient receptor potential-like (TRPL) are Ca(2+)-permeable cation channels found in Drosophila photoreceptor cells associated with large multimeric signaling complexes held together by the scaffolding protein, INAD. To identify novel proteins involved in channel regulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone showed it to be identical to the Drosophila homolog of human FK506-binding protein, FKBP52 (previously known as FKBP59). To determine the function of dFKBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca(2+) influx via TRPL in fura-2 assays. Likewise, purified recombinant dFKBP59 produced a graded inhibition of TRPL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates using recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunoprecipitation resulted in a temperature-dependent dissociation of dFKBP59 and TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lysates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in vivo. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lysates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutagenesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709) eliminated interaction of the TRPL with dFKBP59. These results provide strong support for the hypothesis that immunophilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an important role in modulation of channel activity via interaction with conserved leucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel.

• Parker JS, Mizuguchi K, Gay NJ
• A family of proteins related to Spatzle, the toll receptor ligand, are encoded in the Drosophila genome.
• Proteins. 2001; 45: 71-80
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• The Drosophila gene Spatzle encodes the activating ligand for the Toll receptor. This signaling pathway is required for dorso-ventral patterning in the early embryo and an antifungal immune response in larvae and adults. The genome sequence of Drosophila shows that there are a total of eight Toll-like receptors and these may function in other aspects of embryonic development and innate immunity. Here we describe five Drosophila homologues of Spatzle (Spz2-6) found using an iterative searching method. All five appear to encode proteins containing neurotrophin-like cystine-knot domains. In addition, most retain a characteristic intron-exon structure shared with the prototype Spatzle gene. This provides evidence that the family arose by ancient gene duplication events and indicates that the gene products may represent activating ligands for corresponding Toll receptors. Expression studies show that only Spz4 is expressed strongly in larvae and adults and thus may be involved in an ancillary antifungal response mediated by Toll-5. By contrast, Spz6 shows a complex spatial and temporally regulated expression pattern in the late embryo. Thus the new Toll/Spatzle families of signaling molecules may have important roles in other aspects of development and immunity.

• Davis RL, Turner DL
• Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning.
• Oncogene. 2001; 20: 8342-57
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• The basic-helix-loop-helix (bHLH) proteins are a superfamily of DNA-binding transcription factors that regulate numerous biological processes in both invertebrates and vertebrates. One family of bHLH transcriptional repressors is related to the Drosophila hairy and Enhancer-of-split proteins. These repressors contain a tandem arrangement of the bHLH domain and an adjacent sequence known as the Orange domain, so we refer to these proteins as bHLH-Orange or bHLH-O proteins. Phylogenetic analysis reveals the existence of four bHLH-O subfamilies, with distinct, evolutionarily conserved features. A principal function of bHLH-O proteins is to bind to specific DNA sequences and recruit transcriptional corepressors to inhibit target gene expression. However, it is likely that bHLH-O proteins repress transcription by additional mechanisms as well. Many vertebrate bHLH-O proteins are effectors of the Notch signaling pathway, and bHLH-O proteins are involved in regulating neurogenesis, vasculogenesis, mesoderm segmentation, myogenesis, and T lymphocyte development. In this review, we discuss mechanisms of action and biological roles for the vertebrate bHLH-O proteins, as well as some of the unresolved questions about the functions and regulation of these proteins during development and in human disease.

• Bhaskar V, Valentine SA, Courey AJ
• A functional interaction between dorsal and components of the Smt3 conjugation machinery.
• J Biol Chem. 2000; 275: 4033-40
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• To identify proteins that regulate the function of Dorsal, a Drosophila Rel family transcription factor, we employed a yeast two-hybrid screen to search for genes encoding Dorsal-interacting proteins. Six genes were identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus), three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9), a protein thought to conjugate the ubiquitin-like polypeptide Smt3 to protein substrates. We have found that DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 was found to relieve inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity was potentiated by the overexpression of DmSmt3. We have also identified a DmSmt3-activating enzyme, DmSAE1/DmSAE2 and found that it further potentiates Dorsal-mediated activation.

• Wang W, Lo P, Frasch M, Lufkin T
• Hmx: an evolutionary conserved homeobox gene family expressed in the developing nervous system in mice and Drosophila.
• Mech Dev. 2000; 99: 123-37
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• Three homeobox genes, one from Drosophila melanogaster (Drosophila Hmx gene) and two from mouse (murine Hmx2 and Hmx3) were isolated and the full-length cDNAs and corresponding genomic structures were characterized. The striking homeodomain similarity encoded by these three genes to previously identified genes in sea urchin, chick and human, as well as the recently cloned murine Hmx1 gene, and the low homology to other homeobox genes indicate that the Hmx genes comprise a novel gene family. The widespread existence of Hmx genes in the animal kingdom suggests that this gene family is of ancient origin. Drosophila Hmx was mapped to the 90B5 region of Chromosome 3 and at early embryonic stages is primarily expressed in distinct areas of the neuroectoderm and subsets of neuroblasts in the developing fly brain. Later its expression continues in rostral areas of the brain in a segmented pattern, suggesting a putative role in the development of the Drosophila central nervous system. During evolution, mouse Hmx2 and Hmx3 may have retained a primary function in central nervous system development as suggested by their expression in the postmitotic cells of the neural tube, as well as in the hypothalamus, the mesencephalon, metencephalon and discrete regions in the myelencephalon during embryogenesis. Hmx1 has diverged from other Hmx members by its expression in the dorsal root, sympathetic and vagal nerve (X) ganglia. Aside from their expression in the developing nervous system, all three Hmx genes display expression in sensory organ development, and in the adult uterus. Hmx2 and Hmx3 show identical expression in the otic vesicle, whereas Hmx1 is strongly expressed in the developing eye. Transgenic mouse lines were generated to examine the DNA regulatory elements controlling Hmx2 and Hmx3. Transgenic constructs spanning more than 31 kb of genomic DNA gave reproducible expression patterns in the developing central and peripheral nervous systems, eye, ear and other tissues, yet failed to fully recapitulate the endogenous expression pattern of either Hmx2 or Hmx3, suggesting both the presence and absence of certain critical enhancers in the transgenes, or the requirement of proximal enhancers to work synergistically.

• Drier EA, Govind S, Steward R
• Cactus-independent regulation of Dorsal nuclear import by the ventral signal.
• Curr Biol. 2000; 10: 23-6
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• Rel-family transcription factors function in a variety of biological processes, including development and immunity. During early Drosophila development, the Toll-Cactus-Dorsal pathway regulates the establishment of the embryonic dorsoventral axis. The last step in this pathway is the graded nuclear import of the Rel protein Dorsal. Dorsal is retained in the cytoplasm by the IkappaB-family protein Cactus. Phosphorylation of both Dorsal and Cactus is regulated by a Toll-receptor-dependent ventral signal relayed by the Tube and Pelle proteins. Phosphorylation of Cactus leads to its degradation and to the release of Dorsal to form a ventral-to-dorsal nuclear Dorsal gradient. To understand how the ventral signal regulates the nuclear import and activity of Dorsal, we deleted its conserved nuclear localization signal (NLS). The truncated protein remained in the cytoplasm and could antagonize the function of wild-type Dorsal, suggesting that Dorsal forms a dimer in the cytoplasm. Further, the nuclear import of a mutant Dorsal protein that failed to interact with Cactus was still regulated by the ventral signal. Our results are consistent with a model in which ventral signal-dependent modification of both Cactus and Dorsal is required for the graded nuclear import of Dorsal.

• Espinas ML, Canudas S, Fanti L, Pimpinelli S, Casanova J, Azorin F
• The GAGA factor of Drosophila interacts with SAP18, a Sin3-associated polypeptide.
• EMBO Rep. 2000; 1: 253-9
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• SAP18, a polypeptide associated with the Sin3-HDAC co-repressor complex, was identified in a yeast two-hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S-transferase pull-down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, dSAP18 and GAGA co-localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the dSAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl67 show the homeotic transformation of segment A6 into A5, indicating that GAGA-dSAP18 interaction contributes to the functional regulation of the iab-6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3-HDAC complex, GAGA might contribute to the regulation of homeotic gene expression.

• Dockendorff TC, Tang Z, Jongens TA
• Cloning of karyopherin-alpha3 from Drosophila through its interaction with the nuclear localization sequence of germ cell-less protein.
• Biol Chem. 1999; 380: 1263-72
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• The D. melanogaster germ cell-less (gcl) gene has previously been shown to play a key role in the establishment of the germ cell lineage during fly embryogenesis. To identify other molecules that function with Gcl in this process, we have conducted a yeast two-hybrid screen that utilized Gcl protein as bait. A predominant class of Gcl-interacting clones encodes a species of importin-alpha from Drosophila (karyopherin-alpha3; kap-alpha3), a nuclear-localization sequence binding protein previously shown to act in the transport of proteins from the cytoplasm to the nucleus. The expression of kap-alpha3 is widespread both temporally and spatially throughout the embryo during development, as judged by Northern blotting and whole-mount in situ hybridization to Drosophila embryos, suggesting that it functions at multiple stages of development. Studies of the Gcl/Kap-alpha3 interaction have identified a functional nuclear-localization sequence in Gcl protein which is necessary for an in vivo interaction and for nuclear entry of Gcl, making it likely that one role for Kap-alpha3 is to deliver Gcl protein to the nucleus. The identification of Kap-alpha3 and an in vivo substrate will allow for further characterization of the basis for specificity between importin-alpha molecules and their binding substrates.

• Tefft JD et al.
• Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis.
• Curr Biol. 1999; 9: 219-22
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• In Drosophila embryos, the loss of sprouty gene function enhances branching of the respiratory system. Three human sprouty homologues (h-Spry1-3) have been cloned recently, but their function is as yet unknown [1]. Here, we show that a murine sprouty gene (mSpry-2), the product of which shares 97% homology with the respective human protein, is expressed in the embryonic murine lung. We used an antisense oligonucleotide strategy to reduce expression of mSpry-2 by 96%, as measured by competitive reverse transcriptase PCR, in E11. 5 murine embryonic lungs cultured for 4 days [2]. Morphologically, the decrease in mSpry-2 expression resulted in a 72% increase in embryonic murine lung branching morphogenesis as well as a significant increase in expression of the lung epithelial marker genes SP-C, SP-B and SP-A. These results support a striking conservation of function between the Drosophila and mammalian sprouty gene families to negatively modulate respiratory organogenesis.

• Bohni R et al.
• Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4.
• Cell. 1999; 97: 865-75
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• The control of growth is fundamental to the developing metazoan. Here, we show that CHICO, a Drosophila homolog of vertebrate IRS1-4, plays an essential role in the control of cell size and growth. Animals mutant for chico are less than half the size of wild-type flies, owing to fewer and smaller cells. In mosaic animals, chico homozygous cells grow slower than their heterozygous siblings, show an autonomous reduction in cell size, and form organs of reduced size. Although chico flies are smaller, they show an almost 2-fold increase in lipid levels. The similarities of the growth defects caused by mutations in chico and the insulin receptor gene in Drosophila and by perturbations of the insulin/IGF1 signaling pathway in vertebrates suggest that this pathway plays a conserved role in the regulation of overall growth by controling cell size, cell number, and metabolism.

• Maurel-Zaffran C, Chauvet S, Jullien N, Miassod R, Pradel J, Aragnol D
• nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis.
• Mech Dev. 1999; 86: 159-63
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• From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.

• Cantera R, Kozlova T, Barillas-Mury C, Kafatos FC
• Muscle structure and innervation are affected by loss of Dorsal in the fruit fly, Drosophila melanogaster.
• Mol Cell Neurosci. 1999; 13: 131-41
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• In Drosophila, the Rel-protein Dorsal and its inhibitor, Cactus, act in signal transduction pathways that control the establishment of dorsoventral polarity during embryogenesis and the immune response during postembryonic life. Here we present data indicating that Dorsal is also involved in the control of development and maintenance of innervation in somatic muscles. Dorsal and Cactus are colocalized in all somatic muscles during postembryonic development. In larvae and adults, these proteins are distributed at low levels in the cytoplasm and nuclei and at much higher levels in the postsynaptic component of glutamatergic neuromuscular junctions. Absence of Dorsal, in homozygous dorsal mutant larvae results in muscle misinsertions, duplications, nuclear hypotrophy, disorganization of actin bundles, and altered subcellular distribution of Cactus. Some muscles show very abnormal neuromuscular junctions, and some motor axon terminals are transformed into growth cone-like structures embedded in synaptotagmin-enriched vesicles. The detailed phenotype suggests a role of Dorsal signalling in the maintenance and plasticity of the NMJ.

• Zinke I, Kirchner C, Chao LC, Tetzlaff MT, Pankratz MJ
• Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system.
• Development. 1999; 126: 5275-84
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• We have isolated a Drosophila mutant, named pumpless, which is defective in food intake and growth at the larval stage. pumpless larvae can initially feed normally upon hatching. However, during late first instar stage, they fail to pump the food from the pharynx into the esophagus and concurrently begin moving away from the food source. Although pumpless larvae do not feed, they do not show the typical physiologic response of starving animals, such as upregulating genes involved in gluconeogenesis or lipid breakdown. The pumpless gene is expressed specifically in the fat body and encodes a protein with homology to a vertebrate enzyme involved in glycine catabolism. Feeding wild-type larvae high levels of amino acids could phenocopy the feeding and growth defects of pumpless mutants. Our data suggest the existence of an amino acid-dependent signal arising from the fat body that induces cessation of feeding in the larva. This signaling system may also mediate growth transition from larval to the pupal stage during Drosophila development.

• Qiu P, Pan PC, Govind S
• A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis.
• Development. 1998; 125: 1909-20
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• In the Drosophila larva, blood cells or hemocytes are formed in the lymph gland. The major blood cell type, called plasmatocyte, is small, non-adhesive and phagocytic. Plasmatocytes differentiate into adhesive lamellocytes to form multilayered capsules around foreign substances or, in mutant melanotic tumor strains, around self tissue. Mutations in cactus or Toll, or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. As maternally encoded proteins, Toll, Cactus and Dorsal, along with Tube and Pelle, participate in a common signal transduction pathway to specify the embryonic dorsal-ventral axis. Using the maternal pathway as a paradigm, we investigated if these proteins have additional roles in larval hemocyte formation and differentiation. Analysis of cactus mutants that lack Cactus protein revealed that almost all of these animals have an overabundance of hemocytes, carry melanotic capsules and die before reaching pupal stages. In addition, the lymph glands of cactus larvae are considerably enlarged. The number of mitotic cells in the cactus and TollD hemolymph is higher than that in the wild-type hemolymph. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals could be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. Our results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva. These findings are discussed in light of similar hematopoietic functions of Rel/I(kappa)B-family proteins in mice.

• Nicolas E, Reichhart JM, Hoffmann JA, Lemaitre B
• In vivo regulation of the IkappaB homologue cactus during the immune response of Drosophila.
• J Biol Chem. 1998; 273: 10463-9
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• The dorsoventral regulatory gene pathway (spatzle/Toll/cactus) controls the expression of several antimicrobial genes during the immune response of Drosophila. This regulatory cascade shows striking similarities with the cytokine-induced activation cascade of NF-kappaB during the inflammatory response in mammals. Here, we have studied the regulation of the IkappaB homologue Cactus in the fat body during the immune response. We observe that the cactus gene is up-regulated in response to immune challenge. Interestingly, the expression of the cactus gene is controlled by the spatzle/Toll/cactus gene pathway, indicating that the cactus gene is autoregulated. We also show that two Cactus isoforms are expressed in the cytoplasm of fat body cells and that they are rapidly degraded and resynthesized after immune challenge. This degradation is also dependent on the Toll signaling pathway. Altogether, our results underline the striking similarities between the regulation of IkappaB and cactus during the immune response.

• Yang J, Steward R
• A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal.
• Proc Natl Acad Sci U S A. 1997; 94: 14524-9
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• The intracellular part of the Rel signal transduction pathway in Drosophila is encoded by Toll, tube, pelle, dorsal, and cactus, and it functions to form the dorsal-ventral axis in the Drosophila embryo. Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus. Tube and Pelle are required to relay the signal from Toll to the Dorsal-Cactus complex. In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal. We confirmed these interactions in an in vitro binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube-Dorsal interaction is necessary for normal signal transduction. We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction.

• Packman LC, Kubota K, Parker J, Gay NJ
• Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IkappaB homologue cactus.
• FEBS Lett. 1997; 400: 45-50
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• Cactus protein is a Drosophila homologue of the mammalian IkappaB family of cytoplasmic anchor proteins. In unstimulated cells they function to retain rel/NFkappaB transcription factors in the cytoplasm but are rapidly degraded in response to signalling. The destruction of cactus or IkappaBalpha allows the rel/NFkappaB transcription factor to relocalise to the nucleus. Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain. In this paper we show that, like mammalian IkappaBalpha, the PEST domain of cactus is phosphorylated by casein kinase II. We have localised the site of modification to a single residue, Ser468, and find no evidence for additional phosphorylation sites. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis.

• Bergmann A et al.
• A gradient of cytoplasmic Cactus degradation establishes the nuclear localization gradient of the dorsal morphogen in Drosophila.
• Mech Dev. 1996; 60: 109-23
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• Dorsoventral axis formation in the Drosophila embryo is established by a signal transduction pathway that comprises the products of at least 12 maternal genes. Two of these genes, dorsal and cactus, show homology to the mammalian transcription factor NF-kappa B and its inhibitor I kappa B, respectively. As in the case for I kappa B and NF-kappa B, Cactus inhibits Dorsal by retaining it in the cytoplasm. In response to the signal produced and transmitted by the products of the other genes, Dorsal translocates to the nucleus preferentially on the ventral side of the embryo. Here, we show that Cactus forms a cytoplasmic concentration gradient inversely correlated to the nuclear translocation gradient of Dorsal. Deletions of the N-terminus and C-terminus of Cactus reveal that two modes of degradation control cactus activity: signal-induced degradation and signal-independent degradation, respectively. Genetic evidence indicates that degradation of Cactus is required, but not sufficient to translocates Dorsal completely into the nucleus.

• Hawcroft G, Alphey L
• A Drosophila early embryonic ventral transcript encoding a protein phosphatase-1 binding protein.
• Biochem Soc Trans. 1995; 23: 631-631

• Kubota K, Gay NJ
• Calcium destabilises Drosophila cactus protein and dephosphorylates the dorsal transcription factor.
• Biochem Biophys Res Commun. 1995; 214: 1191-6
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• The Drosophila cactus and dorsal proteins are required for the development of embryonic dorso-ventral polarity and probably also for the innate immune response of the insect. Like their mammalian counterparts (the cytoplasmic anchor protein I kappa B and the rel/NF kappa B transcription factors) cactus and dorsal are regulated at the level of nuclear localisation. We showed previously that increased intra-cellular calcium levels induced by the ionophore ionomycin can activate dorsal/cactus complexes in the Drosophila cell line SL2. In order to study further the activation of dorsal/cactus complexes by calcium, we have prepared a cell line (SLDL) in which dorsal is expressed constitutively. In this paper we show that in SLDL cells ionomycin induces a rapid destruction of cactus and dephosphorylation of dorsal. These results suggest a role for the protein phosphatase calcineurin in calcium mediated activation of dorsal/cactus complexes. They also indicate that in the resting cell constitutive phosphorylation of dorsal is in equilibrium with calcium dependent dephosphorylation.

• Tatei K, Levine M
• Specificity of Rel-inhibitor interactions in Drosophila embryos.
• Mol Cell Biol. 1995; 15: 3627-34
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• The Rel family of transcription factors participate in a diverse array of processes, including acute responses to injury and infection, lymphocyte differentiation, and embryonic patterning. These proteins show homology in an extended region spanning about 300 amino acids (the Rel homology domain [RHD]). The RHD mediates both DNA binding and interactions with a family of inhibitor proteins, including I kappa B alpha and cactus. Previous studies have shown that an N-terminal region of the RHD (containing the sequence motif RXXRXRXXC) is important for DNA binding, while the C-terminal nuclear localization sequence is important for inhibitor interactions. Here we present a structure-function analysis of the Drosophila dorsal RHD. These studies identify another sequence within the RHD (region I) that is essential for inhibitor interactions. There is a tight correlation between the conservation of region I sequences and the specificity of Rel-inhibitor interactions in both flies and mammals. Point mutations in the region I sequence can uncouple DNA binding and inhibitor interactions in vitro. The phenotypes associated with the expression of a modified dorsal protein in transgenic Drosophila embryos suggest a similar uncoupling in vivo. Recent crystallographic studies suggest that the region I sequence and the nuclear localization sequence might form a composite surface which interacts with inhibitor proteins.

• Belvin MP, Jin Y, Anderson KV
• Cactus protein degradation mediates Drosophila dorsal-ventral signaling.
• Genes Dev. 1995; 9: 783-93
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• Dorsal-ventral patterning in the Drosophila embryo relies on a signal transduction pathway that is similar to a signaling pathway leading to the activation of the mammalian transcription factor NF-kappa B. Stimulation of this Drosophila pathway on the ventral side of the embryo causes the nuclear translocation of Dorsal, the Drosophila NF-kappa B homolog. Cactus, like its mammalian homolog I kappa B, inhibits nuclear translocation by binding Dorsal and retaining it in the cytoplasm. We show that Cactus, like I kappa B, is rapidly degraded in response to signaling. More importantly, signal-dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to signaling, and that disruption of the Dorsal/Cactus complex is a secondary result of Cactus degradation. Mutant alleles of cactus that encode more stable forms of the protein block signaling, showing that efficient degradation is necessary for signaling. We find that Cactus protein stability is regulated by two independent processes that rely on different regions within the protein: signal-dependent degradation requires sequences in the amino terminus or ankyrin repeats, whereas signal-independent degradation of free Cactus requires the carboxy-terminal region of the protein that includes a PEST sequence.