In the Drosophila melanogaster embryo, dorsoventral polarity is determined by a concentration gradient of the transcription factor Dorsal in the nucleus (1-3). Prior to axis formation, Dorsal is held in the cytoplasm by an inhibitor, Cactus (4-6). During early embryonic development, proteolysis of Cactus in response to a signal transduced on the ventral side of the syncytial embryo releases Dorsal (7-9). Free Dorsal translocates into ventral nuclei, where it directs expression of ventral-specific genes and represses expression of dorsal-specific genes (10). In this manner, the spatially graded disruption of the Dorsal/Cactus complex establishes the dorsoventral body axis (11).
Dorsal and Cactus are translated from messenger RNAs synthesized in the ovary and form a complex distributed uniformly in the unfertilized egg. Following fertilization, 14 rounds of replication and nuclear division occur prior to cellularization, so that axis formation occurs in the context of a syncytium containing hundreds, and eventually thousands, of nuclei. Within this syncytium, spatially regulated protein degradation of Cactus leads to a gradient of Dorsal nuclear localization, with the highest levels of nuclear Dorsal along the ventral midline and lowest levels in the dorsal half of the embryo.
Dorsal and Cactus were identified on the basis of maternal-effect mutations that disrupt embryonic pattern formation (12). Embryos generated by females lacking Dorsal have a dorsalized phenotype; there is no nuclear Dorsal and hence only dorsal-specific tissues are formed. Females homozygous for a mutation that inactivates Cactus generate embryos with an opposite, ventralized phenotype, in which Dorsal enters nuclei on both the ventral and dorsal sides of the embryo (4). Ten additional genes mutate to a dorsalized phenotype; these loci encode components of the signaling pathway that regulates release of Dorsal from the Dorsal/Cactus complex.
The signal transduction pathway that directs the asymmetric nuclear translocation of Dorsal is initiated by Pipe, Nudel, and Windbeutel, the products of three genes expressed in the somatic cells of the ovary (13). A ventrally localized signal is then amplified by an extraembryonic serine proteinase cascade involving the Gastrulation Defective, Snake, and Easter proteins (14-16). The result is a proteolytically activated form of Spatzle, the activating ligand for the transmembrane receptor Toll (17). Transduction of a signal from activated Toll to the Dorsal/Cactus complex requires a scaffolding protein, Tube, and a protein kinase, Pelle (18, 19). Dorsal binds specifically to both Tube and Pelle, which also bind to one another (20, 21). Both Dorsal and Cactus appear to undergo phosphorylation in response to Toll activation (8, 22, 23). Phosphorylation of Cactus is critical for signal transduction; the role of Dorsal phosphorylation in signaling is not yet clear.
Once within ventral nuclei, Dorsal serves to activate expression of ventral-specific genes (eg, twist) and repress expression of dorsal-specific genes, e.g., zen (24). By themselves, Dorsal dimers activate transcription; repression requires binding to a corepressor, the Groucho protein (25). Dorsal function is not uniform across the ventral half of the embryo. Instead, the gradient of nuclear localization is translated as a series of threshold responses into stripes of gene activation and cell fate determination Whereas maternally expressed Dorsal, Cactus, Toll, Tube, and Pelle function in embryonic patterning, zygotically expressed forms of these proteins mediate an immune response in the fat body, the Drosophila organ most similar to the mammalian liver (27). The immune response, which directs expression of peptide antibiotics upon infection, also involves at least two other proteins closely related in sequence to Dorsal. These proteins, DIF (Drosophila immunity factor) and Relish, appear to be regulated in a manner similar to that observed for Dorsal (28, 29).
The immune function of the Dorsal/Cactus complex has been broadly conserved during evolution; NF-kB and IkB, homologues of Dorsal and Cactus, play a critical role in the mammalian acute phase response, as do counterparts of Toll and Pelle (30-32). Activation of NF-kB in response to stimulation of cells with cytokines, such as tumor necrosis factor-a and interleukin-1, leads to activation of genes that regulate both the inflammatory and immune responses. Recent data suggest that the NF-kB/IkB complex, like the Dorsal/Cactus complex, may also function in pattern formation (33, 34).
The central feature of the Dorsal protein is a multifunctional region termed the Rel homology domain. This region mediates the binding of Dorsal to itself, to Cactus, to other specific protein pairing partners, and to DNA (6, 20, 35, 36). The Rel homology domain also contains a nuclear localization signal (NLS) that drives translocation of free Dorsal into the nuclei of the syncytial blastoderm (see Nuclear Import, Export). It is thought that Cactus retains Dorsal outside nuclei, not by actively tethering Dorsal in the cytoplasm, but rather by occluding this NLS and thereby blocking nuclear translocation (36).
The Dorsal/Cactus complex consists of a dimer of the Drosophila transcription factor Dorsal bound to a monomer of its cytoplasmic inhibitor, Cactus (37). Complex formation requires an interaction between the Dorsal Rel domain and a set of six tandemly arrayed motifs in Cactus. These motifs, termed ankyrin repeats, are found among proteins of widely varying function and have been shown to serve as sites of protein-protein interaction. Interaction with Dorsal requires all of the ankyrin repeats in Cactus (5, 6). Carboxy-terminal to the ankyrin repeats is a PEST domain . PEST domains, rich in the amino acid residues proline, glutamine, serine, and threonine, are found in many proteins whose activity is regulated by proteolysis.
Two regions of Cactus govern its susceptibility to degradation (7-9). The PEST domain influences the overall stability of Cactus, whereas a motif amino-terminal to the ankyrin repeats is specifically required for degradation in response to Toll-mediated signaling. The activities of both sites appear to be regulated by phosphorylation (8, 9, 38). Similar sites govern the proteolysis of the IkB proteins, for which it has been directly demonstrated that phosphorylation of serine residues in the amino-terminal motif triggers a rapid and efficient degradation mediated by the proteasome (39-42).
