dorsal
Tube and Pelle are required to relay the signal from Toll to the Dorsal-Cactus
complex. In a yeast two-hybrid assay, Tube and Pelle interact with Dorsal. These interactions have been confirmed 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. A model is presented in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction (Yang, 1997).
The Tube protein, shown previously by genetic studies to act
downstream of Toll, can function in a novel way to enhance DL activity. In the absence of DL, or
when DL is cytoplasmic, Tube is also found in the cytoplasm of transfected cells. But when DL is
localized to the nucleus, so is Tube. Tube can then function to enhance reporter gene expression,
either by cooperation with DL or as a GAL4-tube fusion protein. Tube thus appears capable of
acting both as a chaperon or escort for DL as it moves to the nucleus, and then as a transcriptional
coactivator. The intracytoplasmic domain of Toll, and specifically the region
sharing homology with the interleukin-1 receptor, is sufficient to induce DL-Tube nuclear
translocation (Norris, 1995).
The pelle gene is required for the nuclear import of Dorsal protein (Shelton, 1993).
Pelle has a protein kinase catalytic domain that acts downstream of Tube and both are involved in the destruction of Cactus, allowing for the entry of Dorsal into the nucleus.
Pelle-mediated signaling induces the spatially graded degradation of Cactus. Using a tissue culture system that reconstitutes Pelle-dependent Cactus degradation, it can be shown that a motif in Cactus, resembling the sites of signal-dependent phosphorylation in the vertebrate Cactus homolog IkappaB is essential for Pelle-induced Cactus degradation. Substitution of four serines within this motif with nonphosphorylatable alanine residues generate a mutant Cactus that still functions as a Dorsal inhibitor but is resistant to degradation (Reach, 1996).
Although both Cactus and Dorsal are modified by phosphorylation in response to signaling, it is believed that signal-dependent modification of Cactus precedes modification of Dorsal. Signal-dependent modification of Dorsal presumably occurs downstream of Cactus degradation, since loss of Cactus function induces Dorsal modification in the absence of signaling. Phosphorylation of Dorsal upon release from Cactus could reflect unmasking of a phosphorylation site or an encounter with a protein kinase upon translocation into nuclei (Reach, 1996 and references).
A signaling pathway active on the ventral side of the Drosophila embryo defines dorsoventral polarity. A cell surface signal relayed by Toll, Tube and Pelle releases the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus; free Dorsal translocates into nuclei and directs expression of ventral fates. Using the yeast two-hybrid system and immunoprecipitation experiments, scaffolding and
anchoring interactions were defined among the pathway components. Dorsal binds
specifically to Tube, Pelle and Cactus, and the protein kinase activity of Pelle
differentially regulates its interactions with Dorsal and Tube. Amino acids 47-345 oof Dorsal are sufficient for interaction with both Tube and Pelle. This same region, the Rel homology domain, is also required for dimerization, for DNA binding and for interaction with Cactus. The Dorsal Rel domain is both necessary and sufficient for generation of a dorsoventral nuclear concentration gradient. Pelle and Dorsal interact with two separable domains of Tube. Pelle binds to the amino-terminal region of Tube that spans residues 25 to 173. Dorsal binds a C-terminal domain of Tube (amino acids 257 to 462). This region contains five copies of an evolutionarily conserved, 8-amino-acid repeat and is required for full Tube function. Interaction with Dorsal requires full-length Pelle. In contrast, only about 100 amino acids of Pelle (residues 26 to 129) are necessary and, most likely, sufficient for interaction with Tube. Pelle catalytic activity modulates its interaction with Dorsal and Tube. Drosophila Filamin (an Actin binding protein that localizes to the inner surface of the cell membrane) is identified as a potential adaptor linking the interaction network, via Tube, to
the transmembrane receptor Toll. The Toll/IL-1 receptor homology appears to be both necessary and sufficient for the interaction of Toll with Filamin. The studies reported here have defined minimal interactions for Pelle (residues 26-129) and Tube (residues 25-173) that correspond closely to regions with similarity to a consensus death domain (see Reaper). Death domains have been identified in pathways regulating apoptosis, but their participation in the dorsoventral signaling cascade suggests a more general role in protein interactions mediating signal transduction (Edwards, 1997).
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. Increased intra-cellular calcium levels induced by the ionophore ionomycin
can activate Dorsal/Cactus complexes in the Drosophila cell line SL2. In a cell line in which
dorsal is expressed constitutively, 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 (Kubota, 1995a).
Transgenic females with Dorsal levels roughly twice that of wild-type produce normal
embryos, while a higher level of Dorsal protein results in phenotypes similar to those observed for
loss-of-function cactus mutations. In contrast
to a Dorsal/Cactus ratio of 2.5 which results in fully penetrant weak ventralization, a Cactus/Dorsal
ratio of 3.0 was acceptable by the system. By manipulating Dorsal levels in different Cactus and
dorsal group mutant backgrounds, it was found that the relative amounts of ventral signal to that of the
Dorsal-Cactus complex is important for the elaboration of the normal dorsoventral pattern. In a wild-type embryo, the activities of Dorsal and Cactus are not independently
regulated; excess Cactus is deployed only if a higher level of Dorsal protein is available (Govind, 1993).
Dorsal controls the protein level of Cactus. Additional amounts of Dorsal lead to a corresponding increase in the amount of CACT. Thus, DL directly determines the amount of its inhibitor CACT. CACT in the DL/CACT complex is stabilized, whereas surplus CACT is unstable and gets degraded. This analysis distinguishes two different modes of CACT degradation: signal-induced degradation and single-independent degradation
Due to signal-independent degradation, the level of CACT is down regulated in dl mutant embryos, but it is not completely absent. The ventral signal is present in dl mutants. There is a subtle difference in the abundance of CACT between the dorsal and the ventral side, suggesting that free, uncomplexed CACT is subject to signal-induced degradation on the ventral side. In double dl-pelle mutant flies in which the ventral signal is blocked, there is a significant increase in CACT levels. Deletion of the PEST sequence blocks signal independent degradation of CACT, while N-terminal deletions of CACT block the response of CACT to signal dependent degradation. Complete absence of CACT is not sufficient to translocate DL completely into the nucleus. It is concluded that in addition to signal-induced degradation of CACT, other mechanisms must operate for complete nuclear translocation of DL. Such mechanisms may include some signal input to DL such as phosphorylation of DL by Protein kinase A. Moreover, the existence of CACT/IkappaB homologs that can substitute for lacking CACT cannot be ruled out (Bergmann, 1996).
Three complexes of DL and/or CACT proteins can be detected.
Complex 1 (190 kDa) is a DL protein homodimer (DL2). Complex 2 (270 kDa) consists of one
complex 1 and one CACT molecule (DL2CACT). Complex 3 (200 kDa) is a CACT protein complex that
does not contain DL protein. In wild-type embryos DL2CACT was detected as the major form of DL
protein, and DL2 was minor. Virtually no DL monomer is detected. DL2CACT is a
cytoplasmic form, whereas DL2 is localized mainly in the nuclei, but a small amount of DL2
is also present in the cytoplasm (Isoda, 1994).
The Rel family of transcription factors 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. 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 (Tatei, 1995).
Cactus, a Drosophila homolog of I kappa B, binds to and inhibits Dorsal. Cactus blocks the DNA binding and nuclear localization
functions of Dorsal. In contrast, Dorsal's transcriptional activating region is functional in the
Dorsal-Cactus complex (Lehming, 1995).
Free cytoplasmic Dorsal protein is able to stimulate translation of the Cactus mRNA, either directly or
indirectly. Such an arrangement would enable the
Dorsal protein to be buffered in the cytoplasm of the resting cell over a wide range of
concentrations. Subsequent to biosynthesis the Cactus protein is either
rapidly degraded or incorporated into complexes with Dorsal. Protein that does not associate with
dorsal has a half-life of approximately 40 min whereas that which is incorporated into complexes is
very stable, having a half life in excess of 24 h (Kubota, 1995b).
Dorsal and Cactus are both phosphoproteins that form a stable
cytoplasmic complex. The Cactus protein is stabilized by its interaction with Dorsal. The
Dorsal-Cactus complex dissociates when Dorsal is targeted to the nucleus. While the
phosphorylation of Cactus remains apparently unchanged during early embryogenesis, the
phosphorylation state of Dorsal correlates with its release from Cactus and with its nuclear
localization. This differential phosphorylation event is regulated by the dorsal group pathway (Whalen, 1993).
The Drosophila immune response uses many of the same components as the mammalian innate
immune response, including signaling pathways that activate transcription factors of the
Rel/NK-kappaB family. In response to infection, two Rel proteins, Dif and Dorsal, translocate from the
cytoplasm to the nuclei of larval fat-body cells. The Toll signaling pathway, which controls
dorsal-ventral patterning during Drosophila embryogenesis, regulates the nuclear import of Dorsal in
the immune response, but the Toll pathway is not required for nuclear import of Dif. Dif is properly translocated from fat-body cytoplasm to nuclei in response to infection in Toll and pelle mutant larvae.
Cytoplasmic retention of both Dorsal and Dif depends on Cactus protein; nuclear import of Dorsal and
Dif is accompanied by degradation of Cactus. Therefore the two signaling pathways that target
Cactus for degradation must discriminate between Cactus-Dorsal and Cactus-Dif complexes. New genes have been identified that are required for normal induction of transcription of antibacterial peptide diptericin
during the immune response. Mutations in three of these genes prevent nuclear import of Dif in
response to infection, and define new components of signalling pathways involving Rel. The 18-wheeler gene, which encodes transmembrane protein that is homologous to Toll, is important for the nuclear localization of Dif during the immune response, so two of these genes may encode products that are necessary for 18-wheeler activation or cytoplasmic components that act downstream of 18-wheeler. Mutations in three other genes, constituting a second class of mutations, cause constitutive nuclear localization of Dif; these mutations may block Rel protein
activity by a novel mechanism. The gene immune deficiency (imd) belongs to this second class, as both Dif and Dorsal are constitutively nuclear in imd mutants. Cactus does not appear to be found in the nucleus in class II mutants. One hypothesis that fits these observations is that the class II genes are required to allow the formation of a nuclear complex of Dif with other proteins, and that this complex is required both for activation of diptericin transcription and for turnover of the nuclear Rel proteins (Wu, 1998).
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, its conserved nuclear localization signal
(NLS) was deleted. The truncated protein remains in the cytoplasm and can 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 fails to interact with Cactus is still regulated by the ventral signal. These 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 (Drier, 1999b).
The prevailing model of the control of Rel protein nuclear import postulates that upon signal-dependent phosphorylation, ubiquitination, and degradation of the IkappaB protein, Rel proteins are 'free' to be imported into the nucleus. Dorsal is phosphorylated in a signal-dependent manner while in the cytoplasm, and this
phosphorylation is essential for high levels of nuclear import. The results presented here further support the
conclusion that the ventral signal directly targets Dorsal and regulates its nuclear import. A mutant 'free' Dorsal that
cannot interact with Cactus is targeted to the nucleus at high levels only in the presence of the ventral signal, whereas
in the absence of the signal it is targeted to the nucleus at low levels characteristic of lateral regions (Drier, 1999b).
All that is known so far leads to a two-level model for the regulating Dorsal nuclear import. Without the ventral signal, a Dorsal-Cactus complex consisting of a Dorsal homodimer and a single Cactus
molecule is sequestered in the cytoplasm. This trimeric complex can form even if only one of the two Dorsal subunits
can interact with Cactus, as suggested by the fact that wild-type Dorsal can maintain S234P-Dorsal in the cytoplasm in
the absence of signaling. All levels of Dorsal nuclear import normally require the signal-dependent phosphorylation and
degradation of Cactus. Once freed from Cactus, Dorsal nuclear import can attain lateral levels. This low level of Dorsal
nuclear import is observed in double-mutant backgrounds that disrupt signaling as well as Cactus activity. However, this
import is dependent on signal-independent phosphorylation of Dorsal (Drier, 1999b).
Level-2 Dorsal nuclear import corresponds to the high level normally observed in ventral and ventrolateral nuclei and is
dependent on the ventral signal, which controls Dorsal phosphorylation. This phosphorylation occurs in the cytoplasm,
since it is present on the deltaNLS-Dorsal mutant; and it occurs while bound to Cactus. The strong structural and
functional conservation between the Rel pathways in Drosophila and vertebrates suggests that the mechanisms
controlling nuclear import of Dorsal will apply to the regulation of other members of the Rel family (Drier, 1999b).
Many developmental and physiological responses rely on the selective translocation of transcriptional regulators in and out of the nucleus through the nuclear pores. The Drosophila gene members only (mbo) encodes a nucleoporin homologous to the mammalian Nup88. mbo is associated with an enhancer trap insertion l(3)5043, identified in a screen for P-element strains that express the lacZ marker in distinct subsets of cells of the Drosophila tracheal system.
The phenotypes of mbo mutants and mbo expression during development are cell specific, indicating that the nuclear import capacity of cells is differentially regulated. Using inducible assays for nucleocytoplasmic trafficking, it has been shown that mRNA export and classic NLS-mediated protein import are unaffected in mbo mutants. Instead, mbo is selectively
required for the nuclear import of the yeast transcription factor GAL4 in a subset of the larval tissues. The first endogenous targets of the mbo
nuclear import pathway have been found in the Rel proteins Dorsal and Dif. In mbo mutants the upstream signaling events leading to the degradation of the IB
homolog Cactus are functional, but Dorsal and Dif remain cytoplasmic and the larval immune response is not activated in response to infection. These results
demonstrate that distinct nuclear import events require different nucleoporins in vivo and suggest a regulatory role for mbo in signal transduction (Uv, 2000).
Homozygous embryos from the l(3)5043 P-element strain show
sporadic but distinct defects in the connecting branches of the tracheal network. In this strain the transposon is inserted into the 5'-untranslated part of
mbo, causing pupal lethality in homozygous animals.
To investigate the function of mbo in the trachea, revertants and strong loss-of-function P-element excision mutants were generated
and characterized. Several of the strains established are homozygous
viable, and five strains, including mbo-1 and mbo-2,
are homozygous lethal and fail to complement either one another, the
original P-element mutation or a chromosomal deletion for the region (Uv, 2000).
During embryogenesis, 100 of the ~1600 cells of the tracheal
epithelium mediate the connection of 20 individual metameres to a
network that facilitates respiration during larval life. Every branch fusion event involves two cells, each located
at the tip of the fusing branches. The fusion cells extend elaborate
cytoplasmic processes, form an intracellular lumen, contact each other,
and finally form a continuous bicellular anastomosis connecting the two
branches. These cells selectively express a
set of fusion marker genes, including l(3)5043 (mbo-lacZ). In mbo-1 embryos 20% of the dorsal anastomoses failed to
form, and in mbo larvae the dorsal branches
remain unconnected. The rest of the trachea, including
the terminal branches that derive from cells not expressing the
mbo-lacZ marker and that grow parallel to the fusion sprouts, are
unaffected in mbo mutants. In addition, the
expression of the early fusion cell marker esg-lacZ and other
tracheal cell-specific markers is not altered in mbo mutant
embryos, suggesting that mbo is required in the fusion cells
subsequent to their cell fate specification. The fusion
cells have another important role in mediating the breakage and release
of old tracheal cuticle at each larval molt.
The mbo-lacZ marker is expressed in the fusion cells
throughout larval life, and all mbo larvae show discontinuities in the tracheal cuticle in positions corresponding to
the fusion junctions of the dorsal trunks. Thus, the
cell-specific zygotic expression of mbo-lacZ in the trachea reflects cell-specific requirements in the fusion cells. In the embryo,
mbo-lacZ expression is also prominent in a subset of the cells of the developing CNS, in dynamic stripes of epidermal cells, in
the lymph glands, and in the intestinal tract including the proventriculus and foregut. Because of the abundant distribution of maternally derived gene products during embryogenesis, however, significant differences in the abundance of RNA or protein could not be detected in these tissues (Uv, 2000).
In the larva, mbo-lacZ is expressed in the fat body, the
trachea, the CNS, and the imaginal tissues. Abundant mbo RNA expression can be detected in the proliferating parts of the larval nerve cord, in the optic lobes of the brain, and in the imaginal disks. Accordingly, the size of the CNS and imaginal
disks of third instar mbo mutant larvae is severely reduced. Because the antiserum against the carboxy-terminal part of
Dnup88 is not sensitive enough to detect the protein by in situ
staining in larvae, a second rabbit antiserum was generated against
the amino-terminal part of Dnup88 and used to describe the
distribution of larval Dnup88. In situ staining of wild-type
larvae with this antiserum shows that the distribution of larval
Dnup88 is tissue specific. Nuclear Dnup88 staining could be detected in
the fat body, trachea, CNS, and imaginal disks but not in the
epidermis, muscles, and gut. This tissue-specific staining is absent
in mbo mutant larvae (Uv, 2000).
To identify endogenous targets of the Dnup88 import pathway, the translocation of Drosophila proteins that enter the
nucleus upon signaling during developmental and physiological responses was examined. During early embryogenesis, the Drosophila NF-kappaB protein
Dorsal is released from the IkappaB homolog Cactus in response to
signaling from the Toll receptor and becomes nuclear on the ventral
side of the embryo to activate transcription. The mbo gene product is maternally provided, since early embryos (0-90 min after egg laying) contain both Mbo mRNA and protein. To
study the embryonic function of mbo in animals devoid of the maternal product, attempts were made to generate embryos from mbo homozygous germ-line clones.
Such germ-line clones were, however, unable to produce eggs; and dissection of mosaic ovaries followed by DAPI staining revealed an early mbo function in
oogenesis, because mutant ovarioles do not form an oocyte. In addition, eggs from mothers homozygous for two hypomorphic mbo alleles contain an increased
amount of dorsal appendage material, resembling the mutant phenotypes of genes involved in determining the dorsoventral polarity of the oocyte. Because of the difficulty in generating embryos lacking maternal Dnup88, the functional analysis focussed on early third instar larvae that lack the
zygotic mbo gene product and contain only small residual amounts of maternal Dnup88. The Dorsal signaling cascade is part of the activation of the
larval immune response in the fat body and is induced upon challenging
the larvae with a bacterial infection. The subcellular localization of Dorsal was examined in fat bodies of
homozygous mbo larvae and their heterozygous siblings. When mbo
heterozygous larvae are infected with a needle dipped in a bacterial
culture, Dorsal becomes translocated into the nuclei of fat body cells
within 45 min. In mbo mutants that receive the same
treatment, Dorsal remains cytoplasmic. Dorsal enters the nuclei of the larval hematopoietic organ, the
lymph gland, upon infection of wild-type animals. Also in
this tissue, the nuclear translocation of Dorsal is severely impaired in mbo mutants (Uv, 2000).
Because the basic transport machinery across the nuclear pore appears
functional in mbo mutants it was anticipated that Dnup88 might
participate in a protein complex that facilitates nuclear translocation
of Dorsal. Indeed, Dnup88 is bound by Dorsal, but the fraction of Dorsal
protein found in complex with Dnup88 is smaller than the amount of
Dorsal bound to Cactus. Thus, Dnup88 appears to participate directly in Dorsal nuclear import (Uv, 2000).
In response to a bacterial injection in wild-type larvae, nuclear translocation of the Rel proteins Dorsal and Dif is followed by the rapid transcriptional activation of genes encodingn antimicrobial peptides. Whether the inducible nuclear entry of Dif might also require mbo was examined. Like Dorsal, Dif is translocated into the fat body nuclei of mbo heterozygous larvae upon infection, and this translocation is impaired in mbo larvae. Accordingly, a reporter construct, composed of the inducible cecropin A promoter coupled to lacZ (cecA1-lacZ), is strongly
induced in heterozygous larvae upon infection, whereas it is
nonresponsive in mbo mutants). An analysis of the
inducible expression of the genes for antimicrobial peptides Drosomycin
and Diptericin by Northern blot hybridizations reveals that their
induction is also severely impaired in mbo larvae.
These results indicate that at least two of the identified fly Rel
transcription factors require mbo for their nuclear entry and
the concomitant activation of their target genes during the larval
immune response (Uv, 2000).
To identify proteins that regulate the function of Dorsal, a yeast
two-hybrid screen was used to search for genes encoding Dorsal-interacting proteins. Six genes have been 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: lesswright). The name 'Ubc9' reflects the homology of this protein to ubiquitin-conjugating enzymes.
However, recent studies on yeast and human Ubc9 have shown that this enzyme primarily conjugates the yeast protein Smt3p or its human homologs SMT3A,
SMT3B, and SMT3C rather than ubiquitin to proteins. DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells,
DmUbc9 relieves inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9
on Dorsal activity is potentiated by the overexpression of DmSmt3. A DmSmt3-activating enzyme, DmSAE1/DmSAE2, has been identified, and found to
further potentiate Dorsal-mediated activation (Bhaskar, 2000).
Smt3 homologs have been
cloned from eukaryotes as diverse as yeast, Arabidopsis, and
humans. In general, these proteins display greater than
50% identity with one another but also roughly 20% identity with
ubiquitin. The identification of the components of the Smt3 conjugation
pathway in yeast, humans, and now Drosophila has revealed
that Smt3 conjugation and ubiquitin conjugation proceed by similar
pathways. Both pathways require an activating enzyme, or
E1 protein, which becomes covalently attached to ubiquitin or Smt3 via
a high energy thioester bond, and a conjugating enzyme, or E2 protein,
which accepts ubiquitin or Smt3 from the E1 protein forming a second
thioester-linked covalent complex. Ubiquitin or Smt3 is then
transferred to an epsilon-amino group on a final protein substrate. The
transfer of ubiquitin from the E2 protein to the final substrate often
requires a ubiquitin ligase, or E3 protein. In contrast, an E3-type
protein is apparently not required for Smt3 conjugation (Bhaskar, 2000 and references therein).
Although ubiquitin conjugation targets proteins for proteasomal
degradation, Smt3 conjugation appears to serve other purposes.
Originally identified in yeast as an enzyme required for proper cell
cycle progression, Ubc9 has been found to physically interact with a
diverse array of proteins, including RanGAP1, PML (promyelocytic leukemia protein),
bleomycin hydrolase, E2A, androgen receptor, and c-Rel. Association of human Ubc9 with RanGAP1 results in the
conjugation of RanGAP1 to the Smt3 homolog SMT3C/SUMO-1
(small ubiquitin-related modifier), allowing it to bind RanBP2 at the nuclear periphery. This allows RanGAP1 to stimulate GTP hydrolysis by Ran. Only SUMO-1-conjugated RanGAP1 binds to RanBP2, implying that SMT3C and Ubc9 are required for
nuclear import. In the case of PML, interaction with Ubc9 and
subsequent SUMO-1 conjugation is essential for targeting PML to
discreet subnuclear structures known as PML-bodies or nuclear dots. In
acute promyelocytic leukemia cells, the subnuclear localization of PML
is altered, suggesting that improper SUMO-1 conjugation may trigger
oncogenesis. These studies argue that one function of Smt3 conjugation
is to regulate the subcellular localization of proteins (Bhaskar, 2000 and references therein).
Although Smt3
conjugation may play a role in regulating Dorsal activity, a number of reports have
implicated Ubc9 in the modulation of transcriptional activation by
other Rel family proteins. For example, SUMO-1-conjugated IkappaB is resistant to degradation and, accordingly,
SUMO-1 and Ubc9 work together to inhibit activation of an
NFkappaB-dependent reporter. This contrasts with the current
findings, which show that the Smt3 conjugation pathway activates
Dorsal-dependent reporters. This difference could relate to
inherent differences between the NFkappaB/IkappaB and Dorsal/Cactus
pathways. However, an earlier report suggests that mammalian Ubc9
can enhance Rel protein function via an interaction with NFkappaB and/or
IkappaB. Thus, an alternative explanation for the different effects of
Smt3 conjugation on Rel protein activity could be that different Smt3
family proteins have different functions. An alignment of DmSmt3 with
the three members of the human SMT3 family reveals
that DmSmt3 displays significantly higher homology to SMT3A and SMT3B
(77% and 75%, respectively) than to SMT3C/SUMO-1 (55%). Thus, DmSmt3, SMT3A, and SMT3B appear to define an Smt3 subfamily that is distinct from SMT3C/SUMO-1. Perhaps SMT3C/SUMO-1 antagonizes transcriptional activation by Rel proteins, whereas SMT3A/B-like proteins (such as
DmSmt3) enhance Rel protein function (Bhaskar, 2000 and references therein).
The Smt3 conjugation system may also function at other levels in the
regulation of Rel family protein activity. For example, Ubc9 has been
shown to associate with the type I TNFalpha receptor and MEKK1 and to
synergize with MEKK1 to activate an NFkappaB-dependent reporter. Although no DmSmt3-Dorsal conjugate could be detected in cells that
were simultaneously co-transfected with Dorsal, DmUbc9, and DmSmt3, the
level of conjugation is low: no more than about 10% of the Dorsal
protein is found in the DmSmt3-conjugated form. Perhaps the
conjugation of DmSmt3 to Dorsal is transient. Perhaps Dorsal and
DmSmt3 are deconjugated as soon as Dorsal enters the nucleus. In accord
with this idea, recent observations suggest that a dynamic equilibrium
may exist between Smt3-conjugated and unconjugated protein species. In
yeast, the vast majority of cellular Smt3p is conjugated to other
proteins, although the population of proteins that is covalently
modified changes during the cell cycle. Furthermore, a yeast enzyme
capable of catalyzing the deconjugation reaction has been identified, and homologs of this enzyme appear to exist in many other eukaryotic species (Bhaskar, 2000 and references therein).
A genetically defined locus, termed semushi (Epps, 1998) is identical with DmUbc9. Experiments employing the
semushi allele suggest that DmUbc9 may be necessary for the
nuclear import of the anteroposterior patterning morphogen Bicoid.
Embryos lacking maternally supplied DmUbc9 have multiple patterning defects of varying
penetrance. Because of the
complex nature of these defects, their characterization will require
extensive phenotypic analysis and the generation of additional DmUbc9
alleles. The possibility that DmUbc9 has pleiotropic developmental
roles is not surprising given increasing evidence for wide spread roles
of Smt3 conjugation in transcription factor function and in the
targeting of proteins to their proper subcellular locales (Bhaskar, 2000 and references therein).
A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. The Drosophila rel family factor Dorsal is a substrate for Smt3 conjugation. The conjugation machinery enhances Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, the role of Smt3 conjugation in the immune response was investigated. The conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo (Bhaskar, 2002).
Recent results have demonstrated the critical role of the mammalian p62-atypical protein kinase C (aPKC) complex in the activation of NF-kappaB in response to different stimuli. Using the RNA interference technique on Schneider cells it has been shown that Drosophila aPKC (DaPKC) is required for the stimulation of the Toll-signaling pathway, which activates the NF-kappaB homologs Dif and Dorsal. However, DaPKC does not appear to be important for the other Drosophila NF-kappaB signaling cascade, which activates the NF-kappaB homolog Relish in response to lipopolysaccharides. Interestingly, DaPKC functions downstream of the nuclear translocation of Dorsal or Dif, controlling the transcriptional activity of the Drosomycin promoter. The Drosophila Ref(2)P protein is the homolog of mammalian p62, since it binds to DaPKC: its overexpression is sufficient to activate the Drosomycin but not the Attacin promoter, and its depletion severely impairs Toll signaling. Collectively, these results demonstrate the conservation of the p62-aPKC complex for the control of innate immunity signal transduction in Drosophila melanogaster (Avila, 2002).
Drosophila represents an ideal system in which to determine the primary role of the aPKCs in NF-kappaB signal transduction because it encodes only one aPKC isoform. According to the data presented in this study, aPKC is selectively required for the innate immune Toll-signaling pathway, acting downstream of the translocation of Dorsal and Dif and playing a critical role in the induction (a typical NF-kappaB-dependent process) of the antimicrobial peptide gene for Drosomycin. Therefore, it can be argued that the primary role of the aPKCs, particularly that of zetaPKC in higher eukaryotic cells, is to somehow control the transcriptional activity of NF-kappaB through a still not completely understood mechanism that most likely involves the direct phosphorylation of RelA and Dif. Interestingly, in Drosophila it is well documented that the phosphorylation of Dorsal is required not only for its transcriptional activity but also for its nuclear translocation. In Drosophila aPKC-depleted cells, a strong inhibition of Dorsal or Dif nuclear translocation is not observed, suggesting that the role of Drosophila aPKC is independent of the previously characterized role for Dorsal phosphorylation in regulating nuclear translocation. Based on experiments in mammalian systems, which demonstrate that p65 transcriptional activity must be stimulated by phosphorylation, it is possible that the residues that control the transcriptional activities of both Dorsal and Dif are different from those controlling the nuclear import of the protein. It is also possible that Drosophila aPKC-mediated phosphorylation has a subtle, yet important, role in the nuclear translocation of Dif and/or Dorsal. Future studies will address this important issue (Avila, 2002).
These studies also demonstrate that Ref(2)P is most likely the functional homolog of p62 in Drosophila. Like p62, Ref(2)P interacts physically with the aPKCs. Therefore, it appears that the p62-aPKC signaling module, like the Par/aPKC complex, is highly conserved. Importantly, a functional role of Ref(2)P in Toll signaling is demonstrated. Thus, the ectopic expression of Ref(2)P is capable by itself of activating the Drosomycin promoter. More interestingly, its depletion severely impairs the Toll pathway (Drosomycin induction) but not the LPS pathway (Attacin induction). Thus, the Ref(2)P/DaPKC complex is critical for Toll signaling (Avila, 2002).
The results presented here also demonstrate that, similar to the p62-TRAF6 connection in mammals, Ref(2)P and Drosophila TRAF2 physically and functionally interact. Together with the results demonstrating that Drosophila aPKC and Ref(2)P are essential for a downstream event in the Toll-signaling pathway, this suggests that a putative Ref(2)P/aPKC/TRAF2 complex might function in the signal-induced stimulation of Dif or Dorsal transcriptional activity. In this regard, it is noteworthy that recent results suggest that TRAF6, in addition to its role in IKK recruitment and activation, may also be involved in the control of RelA transcriptional activity. However, the role of Drosophila TRAF2 in Toll signaling requires further investigation, since the effect of inhibiting (or mutating) TRAF2 has not yet been reported. Further studies will also address the precise mechanism whereby aPKC controls the Toll pathway. The data presented here clearly establish the conserved role of the homolog of the p62/aPKC cassette in NF-kappaB signaling in Drosophila (Avila, 2002).
Continued: see Dorsal: Protein Interactions part 2/2
dorsal
:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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