misshapen
Clones of cells in the eye homozygous for misshapen are externally rough; in tangential sections, mutant rhabdomeres appear elongated instead of having a round cross-section. Since few rhabdomeres are visible in more basal sections, it is likely that this change is due to abnormalities in either cell shape or cell orientaion. Many of these photoreceptor nuclei are displaced from their normal positions near the apical and basal surfaces of the retina. The number of photoreceptors within each ommatidium is generally normal, although occasional examples of either extra or missing photoreceptors are seen (Treisman, 1997).
Drosophila embryos mutant for JNK (basket), JNK kinase (hemipterous), or Djun display a dorsal open
phenotype, indicating that activation of this pathway is essential for normal embryonic development. To test whether Msn activates the JNK MAPK module in
Drosophila, it was necessary to determine if embryos zygotically mutant for msn also display a dorsal open
phenotype. Two inversion alleles of msn (msn102 and msn172) were used in this analysis. Embryos homozygous for either msn allele or transheterozygous for the two alleles
display a defect in dorsal closure, resembling embryos zygotically mutant for bsk. The
observed defect in dorsal closure is observed at a frequency of ~15%, which is similar to that found
for bsk1. Expression of a msn cDNA in the epidermis rescues the dorsal closure
defect in msn mutant embryos, allowing survival of all homozygotes to the pupal stage; this
demonstrates that the phenotype is attributable to loss of msn function (Su, 1998).
Evidence that msn and basket
function in the same pathway comes from the observation that some
embryos doubly heterozygous for msn and bsk display
a dorsal open phenotype. About 10% of embryos derived from
a cross between msn/+ and
bsk/+ flies exhibit a dorsal open phenotype. The defect in
dorsal closure in embryos doubly heterozygous for msn and
bsk is not a dominant effect of either gene; a defect in
dorsal closure was very rarely observed when
msn/+ or bsk/+
flies are crossed with wild-type flies. The presence of such
defects in doubly heterozygous flies strongly suggests that the genes function in the same pathway. Moreover, the severity of the phenotype correlates with the strength of the bsk allele with which the
msn/+ flies were crossed. To confirm genetically that msn also functions upstream of hemipterous,
embryos doubly heterozygous for msn and hep were examined. hep is
on the X chromosome, and both maternal and zygotic hep
contribute to dorsal closure. To obtain flies doubly heterozygous for
msn and hep, msn/+ males were
crossed with hepr75/+ females. About 35% of the flies obtained from this cross display a
defect in dorsal closure. This finding is very close to the predicted
frequency of 37% for embryos with a reduction in both the maternal and
zygotic dosage of hep and the zygotic dosage of msn. A defect in dorsal closure is not observed when
hep/+ females are crossed with
+/Y males, indicating that embryos with the dorsal
closure defect carry the msn mutation. Reduced dosage of both
zygotic and maternal hep is required for the zygotic
lethality of heterozygous msn/+ embryos. The viability of msn/+ flies lacking one copy of
zygotic hep is reduced by >80% (Su, 1998).
A test of a constitutively active form of Jra (DJun) determined it could
rescue the dorsal open phenotype in msn mutant embryos. Previous studies have shown that activated Djun rescues the
bsk phenotype, indicating that one of the main functions of
JNK is to phosphorylate and activate Jun. A constitutively active form of Jra
was made by replacing the JNK phosphorylation sites with acidic
residues. To test whether this activated Djun
rescues the dorsal open phenotype in msn mutant embryos, it was
expressed under the control of the hsp70 heat shock
promoter in the msn mutant background.
Expression of activated Djun rescues the dorsal open phenotype in most
of the msn mutant embryos; heat shock decreased the
number of embryos with a dorsal open phenotype from about 50%. In addition,
expression of an activated form of thick veins, tkvQ253D, also rescues the dorsal open phenotype in
msn mutant embryos. GAL4 driven by the ectoderm-specific
promoter at 69B was used to direct the expression of UAS-tkvQ253D in msn mutant embryos. This expression of activated tkv partially rescues
the dorsal open phenotype caused by msn; it also has a
dorsalizing effect on the ventral ectoderm of the embryos related to
the earlier function of dpp in establishing the dorsoventral axis, which served to mark embryos expressing activated tkv. Thus, these findings provide genetic evidence that
msn functions upstream of the JNK MAP kinase module in leading
edge cells (Su, 1998).
Germline clones mutant for msn fail to develop into eggs. msn must therefore function in the oocyte or the nurse cells; both cell types require a functional cytoskeleton in order to transport and localize determinants of positional information (Treisman, 1998).
Misshapen
acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings.
Both msn loss- and gain-of-function mutations result in defective ommatidial polarity and wing hair formation.
Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the
planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This
demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase
family in higher eukaryotes. Msn acts in EPP signaling through the JNK
(Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no
apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein
kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, evidence is provided for an involvement of the related MAP kinases of the p38 subfamily in planar polarity
signaling downstream of Msn (Paricio, 1999).
In the Drosophila eye, EPP is reflected in the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline (the equator). This pattern is generated posterior to the morphogenetic furrow when ommatidial preclusters rotate 90° toward the equator, adopting opposite chirality depending on their dorsal or ventral positions. Polarity defects are manifested in the loss of mirror-image symmetry, with the ommatidia misrotating and adopting random chirality or remaining symmetrical. The gain-of-function dsh phenotype (sev-Dsh) has been successfully used in previous reports to identify new components of the Fz/Dsh planar polarity pathway. This same assay, dominant genetic modification of the sev-Dsh phenotype, was used to screen through a large number of known genes. Among the few mutants that show a specific interaction are two msn alleles. msn102 and msn172 are X-ray-induced inversions with breakpoints in the msn gene. Both loss-of-function alleles of msn act as dominant suppressors of sev-Dsh, comparable to other planar polarity-specific Dsh effectors (Paricio, 1999).
In addition, msn has been isolated in a gain-of-function screen for genes involved in planar polarity generation. Overexpression of genes required in planar polarity signaling at the relevant time often results in defects that are similar to the loss-of-function mutant phenotypes, e.g. with Fz and Dsh. In such a screen, ap-GAL4 flies (ap-GAL4 induces overexpression of the corresponding gene in the notum and the dorsal part of the wing), were crossed to the collection of 2200 E/P lines and the progeny were scored for disarranged microchaetae on the notum. One of the lines isolated in this screen, ep(3)0549, shows an abnormal orientation of the microchaetae similar to phenotypes obtained with ap driven Fz overexpression. Similarly, ap-GAL4, ep(3)0549 flies show typical polarity phenotypes on the dorsal surface of the wing where these are manifest in the presence of multiple wing hairs. In situ hybridization experiments to polytene chromosomes and complementation analyses reveal that the EP-element insertion in line ep(3)0549 is in the msn locus and represents a msn allele. Subsequent sequence analyses confirm that the EP insertion is located 24 bp upstream of the 5'-end of a msn cDNA. Taken together, these results suggest that msn is involved in EPP signaling and possibly acts downstream of Dsh (Paricio, 1999).
To gain further confirmation of the role of Msn in Fz/Dsh-mediated polarity signaling, an in vitro assay was used to determine whether Msn acts downstream of Dsh in JNK pathway activation. Previous experiments have shown that expression of Dsh in NIH 3T3 cells activates JNK and Jun phosphorylation, indicating that Dsh is a potent activator of a Jun-kinase pathway. Using the same assay, it was asked whether co-expression of a dominant-negative (kinase-inactive) Msn protein (DN-Msn) has an effect on Dsh-induced Jun phosphorylation. Significantly, co-expression of DN-Msn in this context causes a dramatic concentration-dependent inhibition of Jun phosphorylation. Taken together with the genetic interactions, these experiments confirm that Msn is acting downstream of Fz/Dsh in planar polarity signaling (Paricio, 1999).
msn mutations affect the morphology of the rhabdomeres in photoreceptors, causing malformed, 'misshapen' rhabdomeres, and also, at lower frequency, the number of photoreceptors. In addition, msn is required for the process of dorsal closure, and embryos mutant for msn display a typical dorsal open phenotype. To analyze its requirements in polarity generation, msn mutant clones in the eye and the wing were examined in detail. A phenotypic analysis of eye clones reveals that msn is required for the generation of planar polarity. msn mutant ommatidia containing the normal complement of photoreceptors are often misrotated and display the wrong chiral form or are symmetrical (non-chiral). To confirm that the polarity defects of msn mutant ommatidia are primary defects, and thus implicate msn in polarity generation, ommatidial polarity was examined in msn mutant clones at the earliest possible stage in third instar larval imaginal discs (when tissue polarity genes are required). Spalt is expressed in the R3/R4 precursor pair for about two columns at this stage. In wild type this reflects the regular arrangement and direction of rotation of the preclusters. In msn mutant tissue, ommatidial rotation, and thus polarity, is randomized (e.g. ommatidia rotate in the opposite direction as their wild-type neighbors) showing that these defects result from an early failure in polarity establishment. Thus in the eye, the msn phenotype (defects in polarity, malformed, misshapen and missing photoreceptors) is very reminiscent of other genes involved in both polarity and terminal photoreceptor differentiation (Paricio, 1999).
The fz gene has been implicated in the specification of the R3 cell within the R3/R4 pair in the process of chirality generation. The mosaic analysis of both loss-of-function and gain-of-function fz alleles has shown that Fz signaling is required in R3 for correct ommatidial chirality generation and also induces R3 fate. The genetic interactions and cell culture experiments have shown that msn acts downstream of Fz/Dsh, and thus it was asked whether msn is also involved in the selection of R3 in analogy to the fz requirement. The genotypic composition of mosaic ommatidial clusters were examined within the R3/R4 pair. This analysis revealed that, as is the case for fz, the msn+ cell has a strong preference for adopting the R3 photoreceptor fate. This can often lead to chirality inversions, where the msn+ R4 precursor adopts the R3 position and displaces the original msn- R3 precursor. In summary, the genetic requirements of msn in single photoreceptors, in particular the R3/R4 pair, are very similar to those of fz (Paricio, 1999).
msn mutant clones in the wing affect the process of hair development and polarity. Phalloidin stainings of msn clones in pupal wings reveal that cells mutant for msn show defects in prehair initiation. These range from a complete failure of actin polymerization in the prehair to approximately wild-type levels of actin. Loss of Misshapen activity specifically affects wing hair actin organization, since adherens junction actin in msn clones appears normal. Although in some mutant cells an actin 'hair' is detected in the pupal wing, often at abnormal positions within the cell, the adult hairs in msn- tissue are either missing, branched or stunted. Some cells that generate stunted hairs initiate them at multiple sites (a typical planar polarity phenotype. These phenotypes are reminiscent of the defects observed either when prehair actin organization is disrupted by dominant-negative Cdc42 or after cytochalasin D treatment of cultured pupal wing discs (Paricio, 1999).
To test whether overexpression of Msn in the eye can cause polarity defects comparable to sev-Fz or sev-Dsh, the msn E/P-line ep(3)0549 and a UAS-msn strain were used, and these were crossed to sev-GAL4. The eyes of the resulting flies (sev>msn) are externally rough and reveal typical polarity defects in tangential sections. Taken together with the genetic requirements and the loss-of-function phenotypes, the suppression of the sev-Dsh genotype and the cell culture experiments, these data demonstrate that Msn acts in the Fz/Dsh-mediated polarity signaling downstream of Dsh (Paricio, 1999).
During Drosophila embryogenesis msn acts upstream of the JNK-type MAPK module in dorsal closure signaling. Several lines of evidence support a function of JNK cascade components in the generation of planar polarity. To confirm the cell culture experiments and to determine whether components of JNK signaling act downstream of msn in polarity signaling in vivo, the gain-of-function eye polarity phenotype of msn (sev>msn) was used to test for dominant interactions with mutations in JNK signaling components. In sev>msn eyes, only 62.6% of the ommatidia are correctly oriented (compared with 100% in wild-type eyes) with the remaining ommatidia showing polarity defects as well as defects in photoreceptor shape and differentiation. Reducing the dosage of known components of the JNK cascade causes a strong dominant suppression of sev>msn. These results are consistent with msn acting upstream of the JNK module in polarity signaling and with the notion that Msn generally acts upstream of JNK-like cascades in higher eukaryotes (Paricio, 1999).
Although there is accumulating evidence that JNK-type MAPK modules are involved in planar polarity signaling, the analysis of mutant clones of either hep or bsk alleles shows no or weak phenotypes in imaginal discs. These observations suggest a high degree of redundancy at this level in the polarity signaling pathway. To address this issue further, a potential involvement of related kinases that could account for the proposed redundancy was examined. The recently described Drosophila kinases, belonging to the JNK/p38 class within the MAPK modules were examined for genetic interactions with the planar polarity phenotypes of sev-Dsh and sev-msn. These are obvious candidates to be cooperating with Hep and Bsk in polarity generation. At the level of Hep/JNKK (an MKK7 homolog), two other MKKs have been reported (DMKK3 and DMKK4). Similarly, at the level of Bsk/JNK, two p38-like kinases were isolated (Dp38a and Dp38b). Since no mutants have yet been isolated for these genes, whether deficiencies removing these kinases would show an interaction with sev-Dsh was examined. DMKK3 maps in the vicinity of hep: deficiencies removing DMKK3, Df(X)G24 and Df(X)H6, also remove hep. These deficiencies show externally a very strong suppression of sev-Dsh with a marked decrease of misrotated ommatidia as observed in tangential sections. Deficiency Df(3R)p13 removes the DMKK4 locus and also dominantly suppresses sev-Dsh. Similarly, deficiencies removing either Dp38a, Df(3L)crb87-4 and Df(3L)crbF89-4, or Dp38b, Df(2L)b80e3 and Df(2L)b87e25, are suppressors of sev-Dsh. Whether the respective deficiencies showed an interaction with sev>msn was also examined, and it was found that all of them act as dominant suppressors of this genotype as well. It is interesting to mention that the Msn-induced defects in rhabdomere morphology are also suppressed by those deficiencies. These interactions suggest that the p38 kinases are redundant with JNK in the context of planar polarity signaling (Paricio, 1999).
The effects of Misshapen overexpression and loss-of-function in the wing suggest that it may represent a branchpoint in the eye and wing polarization pathways. Misshapen overexpression in the wing produces a multiple wing hair phenotype similar to that of the wing-specific tissue polarity genes inturned and fuzzy. In contrast, msn loss-of-function clones have defects similar to those of dominant-negative CDC42 expressing cells; hair actin polymerization is defective and adult hairs are missing or stunted. One explanation of these data is that Misshapen acts through the JNK pathway, as it does in the eye, but that the targets of transcriptional activation are the components needed for hair formation. Excess production of these components may lead to multiple hairs and their loss to missing hairs. This model is not favored because Dishevelled, which acts upstream of Misshapen in this pathway, affects only hair polarity and is not required for hair formation. Furthermore, expression in the wing of a kinase-inactive version of JNK, which acts as a dominant negative, has no effect on hair formation or polarity. The data are more consistent with a model in which localized Misshapen activity directly promotes polarized cytoskeletal reorganization leading to hair formation; excess Misshapen might then expand the region of the cell competent for hair outgrowth. According to this model, activation of the Fz/Dsh signal transduction pathway in the wing would not necessarily increase the absolute level of Misshapen activity, but rather serve to localize Misshapen activity within the cell. It will be interesting to determine whether molecules such as CDC42, Inturned or Fuzzy represent wing-specific targets of Misshapen. Taken together with the phenotypes in the eye and its role in dorsal closure, these observations indicate that Msn has a function in both nuclear signaling and cytoskeletal rearrangements (Paricio, 1999).
Several studies in yeast and mammalian cells indicate that STE20 kinases function upstream of MKKKs regulating JNK. Msn is the Drosophila homolog of mammalian NIK, belonging to the SPS1 family of STE20-like kinases. Based on differences in structure and possible regulation, two subfamilies of STE20 kinases have been described: mammalian and Drosophila PAKs (p21 Activated Kinases), which are activated by binding GTP-bound Cdc42 and Rac and contain N-terminal regulatory and a C-terminal kinase domain. Members of the second subfamily, containing the SPS1 kinase in yeast, do not interact physically with Cdc42 or Rac and contain an N-terminal kinase and a C-terminal regulatory domain. Several members of the SPS1 subfamily have been described in mammals, but only a subset of them, such as GCK, GLK and NIK have been shown to activate JNK. Genetic and biochemical studies have recently demonstrated that Msn can activate the JNK module and is required during dorsal closure. Msn also acts upstream of the JNK module in polarity signaling in the eye and the results reported here support this as a general mechanism. It remains unclear how Msn is linked to the Rho/Rac GTPases. Genetic data argue for Msn acting downstream of RhoA/Rac. However, it has also been suggested that Rac and Msn act in parallel pathways (Paricio, 1999).
Although genetic evidence suggests an involvement of bsk (JNK) and hep (JNKK) in polarity signaling, phenotypic analyses suggest that the JNK module components are highly redundant in this process. In the search for other kinases involved, it was found that deficiencies uncovering the genes encoding the recently described p38 MAP kinases (Dp38a and Dp38b), and the MAP kinase kinases (DMKK3 and DMKK4) dominantly suppress the sev>msn and sev-Dsh phenotypes, suggesting that these proteins also function downstream of Dsh and Msn in polarity signaling. It is interesting to note that all phenotypic defects of sev>Msn were dominantly suppressed by mutations in both components of the JNK and the p38 kinase module. In contrast to these interactions, tissue culture experiments in mammalian cells have shown that NIK overexpression leads to JNK phosphorylation, but no detectable p38 activation was observed. This difference can be explained by cell- and tissue-specific requirements, e.g. in Drosophila during dorsal closure, JNK activation downstream of Msn is not redundant, while redundancy and p38 interactions are observed in polarity signaling. Thus, it is tempting to speculate that both JNK and p38 kinases cooperate in polarity generation (Paricio, 1999).
The reported promiscuity of the kinases at both the MKK and the MAPK levels could account for the redundancy. The Drosophila MKKs and JNK/p38 MAPKs also appear to act (at least partially) on overlapping downstream targets. Whereas DMKK3 appears rather specific for p38 activation (although it activates both p38s), DMKK4 and Hep (the MKK7 cognate) both activate Bsk/JNK. Similarly, Bsk/JNK and both Dp38s can phosphorylate the downstream targets dJun and ATF2. Thus, a potential downstream target can still be phosphorylated when one of the upstream kinases is removed, and likewise for their upstream activators. An even more complicated picture may emerge when all relevant kinases are identified. Other examples of redundancy are described in yeast MAP kinases. Although KSS1 and FUS3 normally have specific roles in different pathways, it has been shown that they are redundant in the process of mating and in this case KSS1 replaces Fus3 when the latter is not present. The isolation and analysis of all the respective kinases and their mutants will be necessary to understand fully the contribution of each single kinase in planar polarity signaling (Paricio, 1999).
misshapen (msn) functions upstream of the c-Jun N-terminal kinase
(JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to
promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the
SH2-SH3 adapter protein Nck. Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck.
dock is required for the correct targeting of photoreceptor axons. A structure-function analysis of Msn has been performed in vivo in
Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of
photoreceptor axons. Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in
a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos,
suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor
axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic
analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two
distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase
family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding
may provide the flexibility that allows it to link to distinct upstream signaling systems (Su, 2000).
While a role for Ste20 kinases in promoting JNK
activation has been previously identified, little is known about their
regulation or about the specific in vivo function of these kinases.
Msn has been shown to
function upstream of the Drosophila JNK, Bsk, to stimulate
dorsal closure of the Drosophila embryo. It is
now shown that Msn requires both intact kinase activity and a C-terminal
regulatory domain conserved in a number of Ste20 kinases of the GCK
family in order to activate JNK in vivo in flies. The previous finding
that the C-terminal regulatory domain of mammalian NIK binds the N-terminal
regulatory domain of the mammalian Ste11 kinase MEKK1 led the authors to propose that the
interaction of the C-terminal domain of NIK with downstream Ste11
kinases (DMKKK) is critical for NIK and other GCK family members to
activate the JNK MAP kinase module. However, studies on
NIK were performed in assays in which NIK protein was expressed at high levels, and under these circumstances, NIK is able to mediate JNK
activation independent of an upstream activating signal. The requirement for both the C-terminal domain and the kinase activity of
Msn to promote dorsal closure indicates that these domains are required
in order for GCK family members to activate JNK in a physiologically
relevant setting and suggests that an unknown Drosophila Ste11 kinase
also couples Msn to JNK activation and dorsal closure (Su, 2000).
In addition to its role in JNK activation and dorsal closure, Msn is
critical for the correct targeting of photoreceptor axons in
Drosophila. Thus, the data indicate that msn is
important in vivo for regulating at least two distinct biological
events: dorsal closure and photoreceptor axon pathfinding.
Interestingly, the upstream molecules that regulate msn in
these two pathways are distinct, since a mutation eliminating the
function of Msn in axon guidance does not affect its activity in dorsal
closure. One molecule that may act upstream of Msn in the pathway
leading to JNK activation and dorsal closure is a DTRAF; DTRAF1 can interact with Msn to activate
the JNK pathway in cell lines (Liu, 1999). Mutation of a PXXP motif
in Msn prevents it from binding to Dock and from rescuing photoreceptor
axon pathfinding, indicating that Dock and/or related SH3
domain-containing molecules may act in concert with Msn in this process (Su, 2000).
The mechanism by which upstream factors regulate
Msn is not known. A common requirement for Msn activation may involve its increased
local concentration. This could occur either by the recruitment of Msn
to phosphotyrosine-containing proteins or by DTRAF1-induced aggregation
of Msn, thereby allowing juxtaposed Msn molecules present in the
complex to transphosphorylate and activate each other. Alternatively,
the finding that deletion of the region between the kinase and
C-terminal domains of Msn leads to its constitutive activation raises
the possibility that upstream signals activate Msn by inducing a
conformational change and/or displacing a negative regulator bound to
this region (Su, 2000).
The ability of axons to make precise connections during development
requires the axonal growth cone, localized to the leading edge of
projecting axons, to interpret multiple guidance cues that ultimately
navigate axons to their destinations. Changes in the
growth cone's actin cytoskeleton and/or the affinity for binding of
the integrins to the matrix are thought to be the key elements whereby
guidance cues regulate the path taken by developing axons. The finding that dock is required for
Drosophila photoreceptor axon guidance and targeting
has provided a starting point for beginning to dissect the intracellular
signaling pathways that are activated at the growth cone to mediate
these guidance cues. Dock is a member of a large family of adapter
proteins consisting essentially of SH2 and SH3 domains, of which the
prototypic member is Grb2. SH2-containing adapter molecules regulates
signaling pathways by coupling catalytic molecules bound to their SH3
domains to phosphotyrosine-containing proteins (Su, 2000).
While a number of proteins that bind the SH3 domains of Nck and Dock
have been identified, which of these serve as targets in vivo has been
difficult to resolve. In contrast to the SH2-SH3 adapter molecule Grb2,
for which interaction with the downstream SH3 binding partner Sos has been
demonstrated using genetic evidence, the physiologically relevant
binding partners for Nck and Dock and the downstream signaling pathways
have only recently begun to be defined. In this regard,
the Ste20 kinase Pak has been shown to interact with Dock, and
expression of a myristylated form of Pak can partially rescue the
dock mutant phenotype (Hing, 1999). It is shown here that Msn
also binds to the SH3 domains of Dock and the amino acids that
mediate this binding are required for the correct targeting of
photoreceptor axons (Su, 2000).
However, these findings do not provide conclusive evidence that
msn functions downstream of dock in photoreceptor
targeting. Rather, they highlight the complex role of
msn in photoreceptor targeting and suggest that unraveling
the exact functions of msn in this process is unlikely to be
simple. For example, it is likely that Msn functions in both
photoreceptor cells and the brain. The severe photoreceptor axon
guidance defects observed when msn mutants are rescued with
UAS-msn(P656A, P659A), defective in binding Dock, are stronger
than those caused by either the absence of msn in the eye or
the complete loss of function of dock. Interaction between
Msn and an SH3 domain-containing protein or proteins other than Dock in
nonphotoreceptor cells, such as those in the brain, is a likely
explanation. Although photoreceptor development in
most of the eye disc is normal when rescue is carried out with
UAS-msn(P656A, P659A), defects in
brain development in these larvae may contribute to the axon guidance
phenotype; an enhancer trap insertion in msn shows
expression in the optic lobes as well as in the eye.
This hypothesis is difficult to test directly, as many aspects of optic
lobe development are directly dependent on retinal innervation. Because
the defects in photoreceptor axonal targeting are specific to a
mutation in a proline motif (proline appears at positions 656 and 650) that matches consensus SH3 binding motifs, this phenotype is probably due, at least in part, to
the loss of interaction of Msn with an SH3 domain-containing protein (Su, 2000).
The finding that the dock phenotype is enhanced by the
presence of msn suggests that the signaling pathways
regulated by msn, which are critical for the correct
targeting of R-cell axons, intersect with the signaling pathways
regulated by dock. However, this interaction does not
clarify whether msn functions on the same pathway as
dock or on a parallel pathway. In addition, expression of a
form of Msn that is constitutively active is not
sufficient to rescue the dock phenotype. One possible
explanation for these data is that msn acts downstream of
dock but is not the only downstream mediator of its
function. The Ste20 kinase Pak has been shown to
interact with Dock, and expression of a myristylated form of Pak can
partially rescue the dock mutant phenotype. Interestingly,
this form of Pak predominantly rescues the expansion of growth cones in
the medulla, a process that does not appear to require msn
function in the photoreceptor axons. It is possible that msn
and Pak mediate separable functions of dock in photoreceptor
cells. An alternative possibility is that the function of Msn expressed
in photoreceptor cells is mediated by the binding of Msn to an SH3
domain-containing protein other than Dock. The difference in the
phenotypes caused by loss of msn and loss of dock
in the photoreceptor axons would support this hypothesis. Mutations in
the gene encoding such a hypothetical protein, which would function on
a pathway parallel to the dock pathway, have yet to be identified (Su, 2000).
While this report was under review, Ruan (1999)
reported a role for msn in photoreceptor axonal targeting
and Dock signaling. However, in contrast to the findings reported here that
msn mutant R1 to R6 axons terminate prematurely, Ruan
reported that the R1 to R6 axons overshoot the lamina and terminate in
the medulla. In addition, they found that overexpression of Msn in
photoreceptor cells in dock mutants reversed the overshoot
of the R1 to R6 axons. These findings and other data led them to
conclude that dock and msn act in the same
pathway. The reason for the discrepancy between the findings reported
here and their results is not clear at present. One possibility is that
the expression of Msn was much higher in the studies by Ruan, enabling them to see rescue of the dock mutant phenotype;
they used an enhancer promoter line containing a UAS element inserted
in the 5' promoter region of msn to overexpress
msn. However, Ruan also found that overexpression of
msn in a wild-type background led to the premature
termination of many R-cell growth cones, essentially the same phenotype
as they observed when msn was overexpressed in
dock mutants; thus, it is not clear that this in fact
constitutes rescue of the dock phenotype. In contrast,
expression of a myristylated form of Pak largely rescues the
dock mutant phenotype without inducing additional defects (Su, 2000).
An attractive hypothesis is that Dock and/or related SH3 domain-containing
molecules function as adapters to couple Msn to tyrosine-phosphorylated
proteins in response to signaling by a receptor tyrosine kinase
localized at the axonal growth cone. Eph receptors, which constitute
the largest family of receptor tyrosine kinases, are good candidates
for receptors that may function at the axonal growth cone to regulate
changes in the actin cytoskeleton and/or adhesion of integrins to the
matrix that ultimately facilitate the correct targeting of retinal
axons. NIK kinase activity
is activated in mammalian cells by the EphB1 and EphB2 receptors
and NIK couples EphB1 to both JNK and integrin activation. However, although a Drosophila Eph receptor
kinase (DEK) is expressed on retinal axons, misexpression and
overexpression of wild-type DEK or a kinase-defective form of DEK do
not affect axonal pathfinding in Drosophila (Su, 2000 and references therein).
The intracellular signals activated downstream of Msn that mediate the
correct pathfinding of photoreceptor axons are not yet known. The
finding that regulation of the actin cytoskeleton is critical for
growth cones to navigate correctly suggests that Msn may control the
targeting of photoreceptor axons by regulating the actin cytoskeleton. The downstream
pathways regulated by Msn are likely to be diverse and will not be
limited to the activation of JNK. This is suggested by the finding that
msn is required for oogenesis, while bsk and hep are not, and that ventral defects can be induced by a
kinase-defective form of Msn, although maternal and zygotic
bsk mutants do not show such a phenotype. It is not thought
that msn directs axonal guidance via activation of the JNK
MAP kinase pathway, because photoreceptor axonal targeting shows only
minor defects (including occasional overshooting of R1 to R6 axons), in
bsk1 mutant clones made in a Minute
background in the eye disc. However, because
bsk1 is not a complete loss-of-function mutant,
these studies cannot definitively rule out a role for JNK. Small clones
with mutations in both hep and the other
Drosophila p38 MAPK kinase encoded by licorne also show an apparent overshoot of R1 to R6 axons, resembling the dock phenotype but not the msn phenotype. However, the
dock phenotype could not be rescued with an activated allele of hep,
indicating that activation of the JNK pathway is not sufficient to
rescue the dock phenotype. While a direct
link between Pak family Ste20 kinases and the actin cytoskeleton has
been shown, a direct link between GCK family Ste20 kinases and the
actin cytoskeleton has not yet been demonstrated. Thus, the ability to use genetics to identify and validate
potential targets of Msn should provide a valuable tool to uncover not
only the relevant biological functions regulated by Ste20 kinases but
also their physiological downstream targets (Su, 2000).
Differentiation of the R7 photoreceptor cell is dependent on the Sevenless
receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein
kinase signaling cascade. Kinase suppressor of ras (Ksr) functions genetically
downstream of Ras1 in this signal transduction cascade. Expression of
dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling,
prevents R7 cell differentiation, and causes a rough eye phenotype. To identify
genes that modulate Ras signaling, a screened was carried out for genes that alter Ras1/Ksr
signaling efficiency when misexpressed. In this screen, three known
genes, Lk6, misshapen, and Akap200, were recovered. Seven previously
undescribed genes were recovered; one encodes a novel rel domain member of the NFAT family, and
six encode novel proteins. These genes may represent new components of the RAS
pathway or components of other signaling pathways that can modulate signaling by
RAS (Huang, 2000).
Overexpression of msn in an sE-KDN background enhances the rough eye phenotype. msn encodes the Drosophila homolog of Nck interacting kinase (NIK), a member of the mammalian SPS1 subfamily of the STE20 kinase family. msn is an essential gene involved in dorsal closure during embryogenesis in Drosophila and mutant clones result in misshapen rhabdomeres (due to defects in polarity, malformed and missing photoreceptors) in the adult eye. STE20, the founding member of the family in yeast, acts in the pheromone signaling pathway and activates the yeast MAPK module. However, the upstream activators of the STE20 pathway are not known. msn, the STE20 homolog in Drosophila, acts upstream of the c-Jun amino-terminal kinase (JNK) MAPK cascade required for dorsal closure and has recently been implicated to act downstream of the Frizzled receptor in the epithelial planar polarity pathway. Published results have suggested that msn, when overexpressed in the eye in an otherwise wild-type background, can generate a rough eye. However, in the experiments described here, no effect on eye morphology was found using sE-GAL4 to drive either UAS-msn or EP(3)0609 and EP(3)0549, the two EP lines upstream of the msn gene. Drosophila Jun has been implicated as a downstream target of both the JNK MAPK and RAS1/MAPK signal transduction pathways by overexpression analysis of dominant-negative mutations; however, no other components of either pathway are shared. If the JNK pathway can partially compensate for the RAS1 pathway in eye development, one would expect that msn overexpression would suppress sE-KDN. The overexpression results from this screen suggest that as a misexpression suppressor of RAS1, msn decreases signaling in the pathway. msn may independently inhibit neuronal cell fate, although there is no previous evidence for this. It is possible that the JNK signaling pathway may compete with the RAS pathway for common components. Alternatively, this interaction may be tissue specific and only uncovered in the eye with overexpression. There is also the possibility that misexpression of msn causes promiscuous signaling through an independent pathway that also affects eye morphology. Although no phenotype is seen when msn is overexpressed in the eye, this situation may sensitize the eye and nonspecifically enhance the sE-KDN phenotype (Huang, 2000 and references therein).
Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).
Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen
Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR>regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).
Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution. Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).
The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).
Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. PINCH (Steamer duck), a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, RSU-1, a regulator of Ras signaling in mammalian cells, has been identified as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding Drosophila RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. These results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development (Kadrmas, 2004).
To determine if PINCH contributes to DC, its localization was examined in stage 14 embryos. PINCH and ß-PS integrin colocalize in both the LE and the amnioserosa, consistent with PINCH's established role as an integrin effector. The amnioserosa is an extraembryonic tissue present on the dorsal surface of the embryo. Since it has been established that coordinated signaling between the amnioserosa and migrating epithelium is key to formation of LE focal complexes, PINCH could exert an effect in the LE epithelium, the amnioserosa, or both tissues. stck homozygous mutant embryos rescued with a PINCH:GFP transgene under the control of the endogenous PINCH promoter display PINCH-GFP at the LE of the advancing epithelial sheets. Within the LE, PINCH is precisely localized to areas of active phosphotyrosine signaling at triangular nodes corresponding to apical adherens junctions (Kadrmas, 2004).
Zygotic stck mutants proceed normally through DC with complete lethality arising at the embryo-to-larval transition. When maternal PINCH contribution is eliminated, only 12% of cuticles have wild-type morphology. Dorsal puckers and dorsal holes characteristic of aberrant DC are observed at a 36% and 23% frequency, respectively, indicating that maternally inherited PINCH is a key contributor to the process of DC. Moreover, in the absence of maternal PINCH, epithelial defects are observed in ventral patterning and head involution, indicating that PINCH may have additional functions in the developing embryo. Cuticles from embryos lacking both maternal and zygotic PINCH have the same array of phenotypes (Kadrmas, 2004).
PINCH is composed of five LIM domains, each of which could serve as a protein-binding interface. The SH2-SH3 adaptor protein, Nck2, has been reported to interact with mammalian PINCH. This association is intriguing because the Drosophila homologue of Nck2, Dreadlocks, interacts directly with Misshapen (Msn), a MAP4K in the JNK signaling cascade. As with other components of the JNK pathway, null mutations in msn result in embryonic lethality due to failure of DC. Although no direct binding of PINCH to Dreadlocks was observed in Drosophila, this study uncovered a link between PINCH's role in DC and the JNK cascade by testing for genetic interaction between stck and msn. Reduction of PINCH protein levels by introduction of a single copy of the loss-of-function allele, stck18, into the msn102 homozygous null background allows partial restoration of DC, suggesting that PINCH functions as a negative regulator of JNK signaling (Kadrmas, 2004).
Puckered (Puc) is a JNK phosphatase whose expression is up-regulated in response to JNK activation, setting up a negative feedback loop. During DC, JNK-regulated expression of a Puc-LacZ fusion reporter is restricted to the LE cells. In embryos lacking maternal PINCH, expression of the Puc-LacZ fusion protein is disorganized and present in an expanded number of cells, including those beyond the LE border. This phenotype is similar to Puc-LacZ expression observed in puc loss-of-function mutants and further supports a role for PINCH in the negative regulation of the JNK cascade (Kadrmas, 2004).
Thorax closure is a post-embryonic developmental process with features common to DC, including migration of epithelial sheets and a dependence on JNK signaling. Within the wing disc, cells of the stalk region are functionally similar to LE cells during DC. These cells comprise the eventual fusion site for adjacent imaginal discs and are active in JNK signaling. Spatially restricted JNK signaling in the stalk of wing disc can be visualized via a Puc-LacZ reporter, and PINCH expression overlaps with Puc-LacZ in this area of active JNK signaling. Therefore, as in DC, PINCH is properly positioned to act as a regulator of the JNK cascade (Kadrmas, 2004).
Although msn null mutations are embryonic lethal due to DC failure, flies homozygous for the hypomorphic allele msn3349 are semi-viable and a large proportion of the eclosing adults have thorax closure defects. These observations underscore the similarities between thorax closure and DC. In a stck18 heterozygous background, a greater percentage of msn3349 homozygotes are able to eclose, supporting the hypothesis that PINCH is a negative regulator of the JNK pathway in both dorsal and thorax closure (Kadrmas, 2004).
Drosophila PINCH was purified in complex with its binding partners using tandem affinity purification (TAP)–tagged PINCH (TAP-PINCH). stck homozygous mutant embryos rescued with a TAP:PINCH transgene driven by the endogenous stck promoter to wild-type levels afford material for purification of soluble, cytoplasmic TAP-PINCH complexes in the absence of endogenous PINCH protein. Three partners that copurified stoichiometrically with TAP-PINCH from embryos, as well as in complex with TAP-PINCH from cultured Drosophila S2R+ cells, were identified by mass spectrometric analysis. Consistent with what is observed in mammalian cells, ILK copurified with PINCH. The Drosophila homologue of the parvin/actopaxin family of proteins, CG32528, is also present in PINCH protein complexes. Parvin is known to bind ILK and actin in mammalian systems, but the isolated Parvin/ILK/PINCH complexes are the first to be described in Drosophila. Additionally, a novel 31-kD protein was identified as Drosophila CG9031. The CG9031 protein is 55% identical and 74% similar to human RSU-1, a leucine-rich repeat containing protein first identified as a suppressor of cell transformation by v-Ras and subsequently implicated in regulation of MAP kinase signaling, specifically the JNK and ERK cascades, when overexpressed in cultured cells. Despite its potent ability to act as a tumor suppressor, little is known about the mechanism of action of RSU-1. Its partnership with the PINCH protein allows placement of RSU-1 in a molecular pathway that is linked to integrins (Kadrmas, 2004).
To assess the specificity and nature of the interaction between PINCH and RSU-1, domain-mapping studies were performed in cell culture and in yeast two-hybrid assays. Drosophila RSU-1 copurifies with full-length His-tagged PINCH, but not with a truncated His-tagged PINCH containing only LIM1–3, confirming the specificity of the interaction and suggesting LIM4 and/or 5 is the site of binding. ILK, which binds LIM1 of PINCH, copurifies with both full-length and the truncated LIM1–3 version of His-tagged PINCH, serving as a positive control. Both PINCH and ILK are copurified with His-tagged RSU-1. Moreover, endogenous PINCH and RSU-1 can be coimmunoprecipitated. The site of RSU-1 binding to PINCH was further mapped using yeast two-hybrid analysis. Only cells expressing LIM5 bait/RSU-1 prey activated all three reporters, indicating LIM5 is the site of RSU-1 binding. Consistent with the view that they interact in vivo, PINCH:GFP and RSU-1 are prominently colocalized at integrin-rich muscle attachment sites in Drosophila embryos (Kadrmas, 2004). Drosophila RSU-1, which displays seven leucine-rich repeats with high sequence similarity to small GTPase regulators, is encoded by the CG9031 locus. A P-element insertion allele was characterized that disrupts the RSU-1 coding sequence. Flies homozygous for this mutation within CG9031 are viable and fertile, and lack RSU-1 protein as indicated by Western analysis with multiple anti-RSU-1 antibodies. PINCH and RSU-1 are both expressed in larval wing discs and similar to stck wing clones, the mutation within CG9031 produces flies with wing blisters at 60% penetrance. These data are consistent with PINCH and RSU-1 acting in concert to support integrin-dependent adhesion. The CG9031 gene was named icarus (ics) after the son of Daedalus who had unstable wings (Kadrmas, 2004).
Although elimination of RSU-1 function does not result in dorsal or thorax closure defects, the role of RSU-1 in these processes was evaluated by testing for genetic interactions between ics and msn. Similar to what occurs with reduction of stck dosage, homozygous mutation of ics suppresses DC defects observed in msn102 mutant embryos. Absence of RSU-1 also increases eclosure rates of msn3349 hypomorphs and completely suppresses the thorax defects present in msn3349 animals, suggesting that like PINCH, RSU-1 can function as a negative regulator of JNK signaling. To confirm that the suppression of msn DC defects by ics mutation is mediated by the JNK signaling cascade, RSU-1 was eliminated in basket (bsk) embryos that lack zygotic JNK, the terminal kinase in this cascade. Homozygous ics mutation suppresses the DC defects of bsk1 mutants, confirming that ics loss-of-function mutations affect DC by influencing the JNK cascade. Moreover, wing discs isolated from ics mutants display a 30% increase in active phospho-JNK relative to wild type, providing direct biochemical confirmation that RSU-1 influences JNK activation state in vivo. Although no localized accumulation of RSU-1 during DC was detected, RSU-1 is readily detected by Western analysis in stage 13 embryos that are undergoing DC. Thus, the temporal pattern of RSU-1 expression is consistent with genetic results that highlight its role in regulation of JNK-dependent morphogenesis (Kadrmas, 2004).
Analysis of PINCH and RSU-1 levels in wild-type versus stck or ics mutant embryos provided insight into the physiological significance of their association. In embryos mutant for both maternal and zygotic stck, RSU-1 is dramatically reduced relative to wild-type levels. Likewise, in ics embryos, PINCH levels are also decreased. These observations suggest that PINCH and RSU-1 are reciprocally dependent on each other for maximal expression and/or stability. The mechanism for coordinate regulation of RSU-1 and PINCH remains to be determined. Because the phenotypes associated with loss of RSU-1 represent a subset of stck phenotypes, the processes disturbed in ics mutants may be exquisitely sensitive to PINCH levels. Alternately, RSU-1 may have functions that are independent of its role in PINCH stabilization (Kadrmas, 2004).
The data are consistent with a model in which PINCH could modulate JNK signaling in two distinct ways. (1) PINCH is present at areas where JNK is active and antagonizes JNK signaling. This behavior is reminiscent of Drosophila Puc, a phosphatase regulator of the JNK cascade that establishes a negative feedback loop. PINCH has no intrinsic catalytic activity, but might recruit proteins that could alter the availability or activity of JNK signaling components. Like Puc, PINCH expression is up-regulated in response to constitutive JNK signaling. Availability of RSU-1 at these sites of active JNK signaling could independently regulate JNK signaling or modulate the effects of PINCH on JNK through regulation of PINCH stability. (2) PINCH and RSU-1 are required for integrin-dependent adhesion. PINCH links integrins to the actin cytoskeleton via ILK and Parvin, and these connections could influence both integrin-dependent adhesion and signaling. Integrin signaling, through a variety of tyrosine kinases and Rac, stimulates the JNK cascade; therefore, PINCH may also exert an influence on JNK signaling via integrin. The current findings illustrate that the cellular concentration of PINCH affects the level of RSU-1 and vice versa. Thus, modulation of the ratio of RSU-1 to PINCH could provide a mechanism to regulate JNK signaling during DC and thorax closure in Drosophila. It is hypothesized that PINCH/RSU-1 complexes fine-tune and integrate the JNK and integrin signaling cascades required during morphogenesis, highlighting the potential role of integrin-associated apical junctional complexes as signal coordination points for epithelial morphogenesis (Kadrmas, 2004).
Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, null and hypomorph alleles of the Drosophila Ral gene have been generated. Ral null animals are not viable. Reduced Ral expression in cells of the sensory organ lineage has no effect on cell division but leads to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggest that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst, a protein complex involved in vesicles trafficking, specifically the tethering and spatial targeting of post-Golgi vesicles to the plasma membrane prior to vesicle fusion, is a Ral effector. Epistasis between mutants of Ral and of misshapen (msn), the fly ortholog of HGK, suggests the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. It is conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. It is proposed that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK (Balakireva, 2006).
The Ral pathway is an essential component of physiological Ras signaling as well as Ras-driven oncogenesis. It can be instrumental in oncogenic transformation, and an activated form of a Ral exchange factor, Rlf, recapitulates the capacity of Ras to transform immortalized human cell cultures, either alone or together with other Ras effectors. Reciprocally, the lack of RalGDS, another Ral exchange factor, reduces tumorigenesis in a multistage skin carcinogenesis model and transformation by Ras in tissue culture. The molecular basis of the Ral contribution to oncogenesis remains to be elucidated (Balakireva, 2006).
None of the Ral effectors and their attributed cellular functions are obvious actors in oncogenesis. One of the two well-documented Ral effectors, RLIP76/RalBP1, is involved in endocytosis. The other, the exocyst complex, is involved in secretion, polarized exocytosis, and migration and can be found at the tip of filopods and at tight junctions. The exocyst complex is composed of eight proteins, which have been initially identified via mutants of secretion in the budding yeast. Exocyst complexes are bound to vesicles and are supposed to participate in vesicle trafficking and tethering to the plasma membrane. Globally, Ral appears to be a regulator of vesicle trafficking with consequences on cell proliferation, cell fate, and cell signaling (Balakireva, 2006).
In order to gain insight into Ral function, a genetic and cell biology approach was undertaken using Drosophila, which has a single Ral gene. Null and hypomorph alleles of Ral were generated, and Ral was shown to be an essential gene. Ral loss-of-function has dramatic effects on the differentiation of sensory organ precursor cells and leads to caspase-8-independent cell death by releasing ectopic tumor necrosis factor (TNF) receptor-associated factor 1-c-Jun N-terminal kinase (TRAF1-JNK) signaling. Sensory organ cell survival in Ral mutants is rescued by an activation of p38 mitogen-activated protein (MAP) kinase, revealing an antiapoptotic function of this latter. The influence of Ral on sensory organ cell fate is directly mediated by the exocyst complex together with a novel interaction partner, the MAP4K4 (also known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase [HGK] in mammals and Misshapen [MSN] in flies). This suggests that a Ral/exocyst/JNK regulatory axis may represent a key component of developmental regulatory programs (Balakireva, 2006).
Hypomorph mutations of Ral displayed a loss-of-bristle phenotype with sockets without shafts, as do flies expressing dominant negative alleles of Ral). Whereas Ral is expressed in many if not all tissues, the only situation where a decreased level of Ral appears compatible with adult viability leads to a developmental phenotype in the bristle sensory organs. In Ral mutants, the pI precursor cells undergo the right number of divisions with a correct timing, but afterward shaft cells die by apoptosis, showing that death hits after cell division and determination has taken place, during the subsequent differentiation stage (Balakireva, 2006).
The various pathways that lead to apoptosis for their interactions with Ral have been explored. The caspase-8-mediated pathway did not contribute to the Ral phenotype, as opposed to a caspase-9-mediated pathway. The JNK pathway, a cascade of four kinases starting with MSN (MAP4K4 or HGK in human), which requires formation of a complex with TRAF1 for its full activity, and ending at the Jun N-terminal kinase, was tested. Puckered is a phosphatase that dephosphorylates and deactivates JNK (Balakireva, 2006).
Loss-of bristle and apoptosis phenotypes due to decrease of Ral signaling were suppressed by down-regulation of the JNK pathway and enhanced by its up-regulation. Symmetrically, a phenotype due to a hyperactivation of the Ral pathway by the overexpression of RalG20V was suppressed and enhanced by enhancing or decreasing JNK signaling, respectively (Balakireva, 2006).
The fact that the enhancement and suppression can be induced by genetic alterations of TRAF and MSN as well as of JNK proteins suggests that Ral is a general negative regulator of this cascade. Dominant negative alleles of transcriptional effectors of the JNK, Jun itself but also Fos, suppress the Ral phenotype, suggesting that Ral regulates transcriptional events involved positively or negatively in apoptosis (Balakireva, 2006).
Down-regulating the JNK pathway is not only suppresses apoptosis in Ral-defective cells but also rescues normal bristle development. Together with data in S2 cells, where Ral behaves also as a negative regulator of JNK in the absence of any cell death (Sawamoto, 1999), the results suggest a functional relationship between Ral and the JNK pathway wherein Ral activation keeps JNK down. Data using activated and dominant negative alleles of Ral in mammalian cell culture support a positive effect of Ral on JNK activation. The source of this discrepancy, which might be due to cell- and/or context-specific interactions of Ral with the JNK pathway, is not understood. However, the current data obtained by RNA interference in HeLa cells are consistent with the fly model (Balakireva, 2006).
Epistatic relationships between Ral and p38 MAP kinase mutants revealed another actor in Ral-dependent apoptosis: the p38 MAP kinase behaves as an antiapoptotic kinase, which could be positively regulated by Ral (Balakireva, 2006).
A control of the basic JNK activity might serve two purposes: (1) it minimizes JNK activity and avoids undesirable cell death in normal conditions; (2) a low level of basal JNK activity allows better differential in activation of JNK when this activation happens in response to stresses that lead eventually to apoptosis (Balakireva, 2006).
The molecular basis of Ral action on the JNK pathway was addressed genetically and biochemically. The model that emerges is that the exocyst complex is the matchmaker between Ral and the JNK pathway, and the simplest interpretation of genetic data is that the exocyst works like a negative regulator of HGK activity. Finally, the exocyst complex was found to bind in vivo to HGK, providing a biochemical basis for the functional effect of Ral on JNK (Balakireva, 2006).
Decreasing the JNK pathway seems to favor the oncogenic capacity of Ras in mouse primary fibroblasts. The current results can explain one of the contributions of the Ral pathway to oncogenesi: cancer cells have to sustain proliferative signals and relieve proapoptotic signals, and Ral via the exocyst complex might be in charge, at least, of this latter task in oncogenesis. Finally, it has been recently shown that the exocyst complex carries enzymatic activities working in the NF-kappaB pathway. These data together with the present report widen the role of the exocyst to functions other than directing vesicle traffic and contributing to exocytosis (Balakireva, 2006).
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