rolled/MAPK
The role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK) has been examined. Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. Using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, it has been demonstrated that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. When terminal activity is progressively removed, there is a corresponding progressive malformation and eventual loss of terminal cuticular structures. The first terminal cuticular elements that are malformed or lost require the highest terminal activation (e.g., the anal tuft and posterior spiracles visualized by the presence of Filzkorper material). The next elements that are malformed or lost require intermediate levels of terminal signal (e.g., the abdominal 8 (A8) denticle belt and the posterior spiracles). Finally, the last elements that are malformed or lost require the lowest levels of terminal activity (e.g., posterior A7). While in the absence of D-raf activity, no activated MAPK (dp-ERK) is observed at the posterior pole. In csw null mutant embryos, where the tll and Hb expression domains are present though mispositioned, reduced levels of dp-ERK reactivity are observed. Collectively, these results reveal that a precise transcriptional response translates into a specific cell identity (Ghiglione, 1999).
Two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. For example, analysis of Torextracellular-Egfrcytoplasmic reveals that the posterior domain of Hunchback does not retract from the posterior pole, but rather remains as a terminal cap. Further, the anterior border of this posterior Hb domain is shifted posteriorly. Altogether, these results illustrate how a gradient of MAPK activity controls differential gene expression and thus, the establishment of various cell fates. The roles of quantitative mechanisms in defining RTK specificity are discussed. It is possible that in some instances, the generation of differing magnitudes of activity from the cytoplasmic domains of specific RTKs might be dependent on the specific affinities of the downstream signal transducers to the receptor. Csw binds through one of its SH2 domains to only one phosphotyrosine on Tor. Perhaps a higher or lower affinity of Csw to this site, or addition of another site that would also engage the second SH2 domain of Csw, would increase or decrease signal output. Presumably, in each individual cell there exists a mechanism built into the enhancer elements of the promoters of both tll and hkb that acts to read directly the magnitude of Tor signaling. In the tll promoter, a Tor-response element that mediates the repression of tll has been identified, indicating that the Tor signal activates tll by a mechanism of derepression. A putative candidate for this repressor activity is encoded by the transcription factor Grainyhead. Grainyhead binds to the Tor-response element and can be directly phosphorylated by MAPK in vitro: a decrease in Gh activity has been shown to cause tll expansion in early embryos. Further, the transcriptional corepressor Groucho is required for terminal patterning. Further characterization of how Gh and/or Gro activities are regulated by activated MAPK should clairify how differing levels of phosphorylation translate into derepression of terminal target genes (Ghiglione, 1999).
A new gene, heartbroken, has been identified that participates in the signaling pathways of both FGF receptors. heartbroken has been cloned and although it appears to be a novel protein, it possesses several sequences characteristic of a signal transduction protein (Vincent, 1998). Mutations in heartbroken are associated with defects in the migration and later specification of mesodermal and tracheal cells. Genetic interaction and epistasis experiments indicate that heartbroken acts downstream of the two FGF receptors, but either upstream of, or parallel to, Ras1. Furthermore, heartbroken is involved in both the Heartless- and Breathless-dependent activation of Mapk. It has been concluded that heartbroken may contribute to the specificity of developmental responses elicited by FGF receptor signaling (Michelson, 1998, and Vincent, 1998).
Rolled MapK is another important component of the Rtk signalling cascade. A monoclonal antibody specific for the dual phosphorylated, activated form of MapK (diphospho-MapK) has recently been shown to be highly effective for monitoring the activity of Rtk pathways during Drosophila development. Using this reagent, high levels of activated MapK were localized to the leading edge of the migrating mesoderm, with much lower levels present at more ventral positions. Activation of MapK is very weakly enhanced in the ventral mesoderm by twi-GAL4-induced expression of a constitutive form of Htl, although the normal gradient of diphospho-MapK expression does not appear to be significantly altered by this manipulation. Activated MapK is completely absent from the early mesoderm of htl mutants, confirming that this mesodermal expression of diphospho-MapK is entirely Htl-dependent. Moreover, no activated MapK is detectable at comparable stages in the mesoderm of hbr mutant embryos. Activated Htl expressed in a null htl mutant generates a low, uniform level of diphospho-MapK throughout the mesoderm. In addition, reduction of hbr function is capable of completely blocking MapK activation by constitutive Htl. These results suggest that hbr acts upstream of MapK in the Htl signal transduction pathway, a hypothesis that is consistent with the findings of the above genetic epistasis experiments (Michelson, 1998).
The critical role phosphatases play in controlling MAPK activity is best illustrated by genetic studies in the fruit fly, where activation of the ERK/MAPK homolog, encoded by the rolled (rl) gene, is required during a number of developmental events. During R7 photoreceptor differentiation, signaling through the RAS1/MAPK pathway acts as a binary switch to trigger only one of five equivalent cells to differentiate as an R7 neuron, while the lack of signal transmission in the other four cells causes them to adopt the nonneuronal cone cell fate. A gain-of-function mutation in rl/MAPK, called Sevenmaker (rlSem/MAPKSem), was identified based on its ability to trigger R7 differentiation in the absence of upstream signaling events. The rlSem mutation, which results from an Asn-for-Asp substitution in the kinase domain, produces dominant phenotypes similar to constitutive-ectopic activation of the RTK. Most notably, rlSem produces ectopic R7 cells, which results in a mild rough eye. It also causes extra wing veins as well as dominant female sterility. The corresponding Asn substitution in mammalian MAPK does not alter the basal kinase activity in tissue culture cells but rather renders the active protein resistant to a variety of phosphatases. A genetic screen to isolate mutations in genes that act positively or negatively downstream of Ras1 and mutations were identified in genes encoding a number of key downstream signaling proteins, for example, RAF, MEK, MAPK, KSR, 14-3-3, and PP2A. One negative regulator of Ras1 signaling, Enhancer of Ras1 2-5 (ER2-5) has now been cloned and characterized; mutations of this gene are homozygous viable but produce ectopic R7 cells and reduced female fertility. This gene, renamed Protein tyrosine phosphatase-ERK/Enhancer of Ras1 (PTP-ER) encodes a novel tyrosine phosphatase that specifically binds to and dephosphorylates Drosophila MAPK, thereby downregulating its kinase activity. PTP-ER is unable to dephosphorylate and downregulate the Sevenmaker mutant form of Drosophila ERK/MAPK, indicating that resistance to PTP-ER partially accounts for the increased activity and dominant phenotypes associated with this mutant form of MAPK. PTP-ER is related to mammalian PCPTP1, LC-PTP/HePTP, and STEP tyrosine phosphatases. PTP-ER mutants produce extra R7 cells and enhance activated Ras1 signaling. Ectopic expression of PTP-ER dramatically inhibits RAS1/MAPK signaling. PTP-ER binds to and inactivates Drosophila ERK/MAPK; however, it is unable to dephosphorylate and downregulate Drosophila MAPKSevenmaker (Karim, 1999).
PTP-ER is novel among the tyrosine-specific phosphatases because of its unusually large catalytic domain; however, no apparent functional significance can be attributed to these insertions. Consequently, bona fide mammalian orthologs of PTP-ER may not be structural homologs containing similar insertions. Within its key catalytic elements, PTP-ER is most similar to members of the PCPTP1, LC-PTP/HePTP, and STEP tyrosine phosphatase families, particularly EC-PTP. Although much of the long N-terminal domain of PTP-ER is novel, it contains several motifs also found in PCPTP1, LC-PTP/HePTP, and STEP phosphatase families. Most notably, PTP-ER has three so-called KIMs, which have been shown to mediate PTP-SL and STEP binding to mammalian ERK. The KIMs are clearly related to the domain of c-Jun (which is a docking site for JNK), the D box of Elk-1 (which acts as a docking site for both ERK and JNK), and the high-affinity docking motif found at the amino termini of MEKs that mediates the binding of MEKs to their cognate MAPKs. All of these docking sites are characterized by a cluster of basic residues separated by 1-5 residues from an (L/I)x(L/I) motif. PTP-ER also has two DEF motifs (FxFP) in its N-terminal domain, which also act as high-affinity MAPK-binding sites and are found in several proteins known to be MAPK substrate proteins. No DEF motifs are found in EC-PTP, HePTP, or STEP. A strong indication that PCPTP1, LC-PTP/HePTP, and STEP might be mammalian orthologs of PTP-ER comes from the observation that, like PTP-ER, PTP-SL (a PCPTP1 family member) binds to and dephosphorylates ERK in vitro and can reduce ERK activity in transfected tissue culture cells (Karim, 1999 and references).
In order to understand the precise role PTP-ER plays in controlling signal output and/or signaling sensitivity, a better understanding is needed of its regulation. PTP-ER is constitutively expressed in Drosophila S2 cells and does not appear to be induced in response to RTK or Ras1 activation. Constitutive expression of PTP-ER may help attenuate signaling following activation; alternatively, it might function to limit the pool of active MAPK in the cytoplasm, thereby making the system sensitive to further stimulation by growth factors. PTP-ER may also regulate MAPK dimerization and its subsequent nuclear localization. The presence of both KIM and DEF motifs in the N-terminal domain of PTP-ER suggests that this domain mediates the specific interaction between PTP-ER and MAPK. The relative contributions of the KIM and DEF sites to binding is unclear. One possibility is that they have distinct functions. For example, the KIM sites might target MAPK to PTP-ER as a substrate while the DEF sites might target PTP-ER to MAPK as a kinase substrate. Alternatively, all of these sites might act together and promote both activities. The N-terminal domain also has a high concentration of potential MAPK phosphorylation sites, particularly within the first 400 residues clustered around the two DEF sites, indicating that this domain likely mediates regulatory phosphorylation of PTP-ER by MAPK. No negative regulatory effect due to MAPK phosphorylation of PTP-ER could be detected; however, the possibility cannot be ruled out that PTP-ER might require MAPK binding and/or phosphorylation for its activation. Indeed, when coexpressed with constitutively active Ras1V12 and MAPK in Sf9 cells, PTP-ER is catalytically active and displays a slower mobility similar to in vitro phosphorylated protein. The MAPK-specific dual-specificity phosphatase, MKP-3, requires a direct interaction with MAPK but not phosphorylation for its phosphatase activity (Karim, 1999 and references).
A growing body of evidence points to a complex network of both dual-specific and now tyrosine-specific phosphatases (both cytoplasmic and nuclear) that bind to and inactivate MAPK and are themselves regulated by MAPK. These phosphatases contribute to the intricate control of signal duration and sensitivity. Further characterization of the regulation and redundancy of MAPK phosphatases should provide a better understanding of their precise roles in modulating signal transmission and output (Karim, 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).
Output from the circadian clock controls rhythmic behavior through poorly understood mechanisms. In Drosophila, null mutations of the neurofibromatosis-1 (Nf1) gene produce abnormalities of circadian rhythms in locomotor activity. Mutant flies show normal oscillations of the clock genes period (per) and timeless (tim) and of their corresponding proteins, but altered oscillations and levels of a clock-controlled reporter. Mitogen-activated protein kinase (MAPK) activity is increased in Nf1 mutants, and the circadian phenotype is rescued by loss-of-function mutations in the Ras/MAPK pathway. Thus, Nf1 signals through Ras/MAPK in Drosophila. Immunohistochemical staining has revealed a circadian oscillation of phospho-MAPK in the vicinity of nerve terminals containing pigment-dispersing factor (PDF), a secreted output from clock cells, suggesting a coupling of PDF to Ras/MAPK signaling (Williams, 2001).
ERK MAP kinase plays a key role in relaying extracellular signals to transcriptional regulation. Because different activity levels or the different duration of ERK activity can elicit distinct responses in one and the same cell, ERK has to be under strict positive and negative control. Although numerous genes acting positively in the ERK signaling pathway have been recovered in genetic screens, mutations in genes encoding negative ERK regulators appear underrepresented. To this end, the dual-specificity phosphatase Mitogen-activated protein kinase phosphatase 3 (DMKP3) was characterized. A novel assay was established to elucidate the substrate preferences of eukaryotic phosphatases in vivo and thereby confirm the specificity of DMKP3 as an ERK phosphatase. The Dmkp3 overexpression phenotype characterized in this assay permitted the isolation of Dmkp3 null mutations. By genetic analysis it has been shown that DMKP3 and the Protein tyrosine phosphatase-ERK/Enhancer of Ras1 (PTP-ER) perform partially redundant functions on the same substrate, ERK. DMKP3 functions autonomously in a subset of photoreceptor progenitor cells in eye imaginal discs. In addition, DMKP3 function appears to be required in surrounding non-neuronal cells for ommatidial patterning and photoreceptor differentiation (Rintelen, 2003).
MAPKs are evolutionarily conserved enzymes in signaling pathways regulating cellular fates and responses to a variety of extracellular signals. Four subgroups of the MAPK family are defined in metazoans -- ERK, JNK, p38 and ERK5. MAPKs are activated in a cascade by phosphorylation of a threonine and a tyrosine residue in the so-called P-loop by dual-specificity kinases, which in turn are substrates of other kinases. This cascade-like arrangement of three kinases is predicted to make the modules sensitive to regulation and to predispose them to mediate switch-like processes (Rintelen, 2003 and references therein).
A switch mechanism requires the possibility to also counteract the stimulatory activity of the dual-specificity MAPK kinases. This is achieved by phosphatases capable of dephosphorylating either the threonine residue or the tyrosine residue [serine/threonine phosphatases (STPs) or protein tyrosine phosphatases (PTPs)], or both [dual-specificity phosphatases (DSPs)]. Since DSPs exhibit a high specificity towards MAP kinases and within those to a subset of the family, they have also been designated MKPs (for MAP kinase phosphatases). DSPs are comprised of an N-terminal CH2 domain (for Cdc25 homology) implicated in substrate binding, which also contains a basic docking site that directly binds to the negatively charged common docking (CD) domain of MAPKs. Upon MAPK binding the phosphatases undergo a conformational transition that stimulates the activity of the C-terminal catalytic domain. The prevalence of this interaction is illustrated by a dominant ERK mutation termed Sevenmaker, which affects the charge of the CD domain such that the physical interaction of ERK with its DSP is greatly impaired. Thereby the phosphatase activity is compromised and ERK kept in an activated state. Flies carrying the dominant Sevenmaker mutation are viable, but display multiple phenotypes characteristic of an overactive RAS pathway, for example rough eyes because of the recruitment of extra photoreceptor cells (Rintelen, 2003 and references therein).
The Drosophila compound eye is composed of approximately 800 ommatidia, each built up of an equivalent of 19 cells, eight of which are neuronal photoreceptor cells. Photoreceptors contain specialized microvillar stacks of membrane termed 'rhabdomeres'. The rhabdomere of the R7 photoreceptor neuron is situated in the center of the ommatidial unit on top of that of the R8 cell. The rhabdomeres of the remaining six outer photoreceptors are arranged such that ommatidia appear in two different chiral forms. Chirality is conveyed by the R3 and R4 cells, which adopt an asymmetrical position within the ommatidium (Rintelen, 2003 and references therein).
Ommatidial patterning starts in an orderly fashion at the posterior border of eye imaginal discs in third-instar larvae. The differentiation process is accompanied by a visible indentation in the epithelium called the 'morphogenetic furrow' that sweeps across the disc. Within the morphogenetic furrow, groups of cells form 'rosette'-like clusters from which cells are singled out by lateral inhibition to become the neuronal R8 photoreceptor cell. This process requires RAS activity but appears to be independent of the receptor tyrosine kinase EGFR. In a stepwise manner, whereby differentiating cells recruit undifferentiated neighbors, the ommatidia are assembled: When the R8 cell is determined it produces the TGFalpha-like EGFR ligand Spitz. Spitz in turn activates EGFR signaling in two adjacent cells and thereby recruits them to the cluster to form the R2/R5 pair. The new cells attract the presumptive R3/R4 pair by a similar mechanism. Initially, one or two additional cells are incorporated into the growing cluster. These so-called mystery cells are expelled from the precluster when the R3/R4 pair differentiates. A gradient in Frizzled activity originating from the dorso-ventral midline of the eye field (equator) generates a difference between the initially equivalent R3 and R4 precursors that is then amplified by a Notch-Delta interaction. The cell closer to the equator will exhibit high Delta levels and will be instructed to become an R3 cell. The more polar cell has high Notch activity and differentiates as R4. Subsequently, the dorsal and ventral preclusters rotate by 90° in opposite directions thereby establishing chirality. Of the last three photoreceptor cells recruited to the precluster, the middle cell chooses the R7 fate and the two others form the R1/R6 pair. In contrast to the R8 cell, the remaining photoreceptors are dependent on high and/or sustained Ras pathway activity. Overactivation of ERK by constitutively active RAS or receptor tyrosine kinases results in severe differentiation defects. This phenotype is mimicked by loss-of-function mutations in negative regulators of the RAS signaling pathway, like Gap1 or the ETS transcriptional inhibitor Yan. Surprisingly, apart from PTP-ER, mutations in genes coding for ERK phosphatases have not been identified based on a similar phenotype. It is thus possible that various phosphatases perform redundant functions on ERK. Redundancy could explain why mutants of the mouse DSP MKP1 and the C. elegans lip-1 are fully viable. Likewise, HE-PTP knockout mice devoid of the ERK tyrosine phosphatase are phenotypically normal and the corresponding Drosophila PTP-ER mutants only exhibit slight defects (Rintelen, 2003 and references therein).
Mammalian dual specificity phosphatases MKP3 and MKP4 and their Drosophila homolog DMKP3 (MKP3 -- FlyBase) selectively inhibit ERK in vivo. Analysis of Dmkp3 loss of function mutations reveals that DMKP3 performs redundant and non-redundant functions on ERK together with the tyrosine-phosphatase PTP-ER. These results further suggest that RAS signaling is not only required within the photoreceptors to properly differentiate, but also performs a function in surrounding cells to shape the developing ommatidium. Together, evidence is provided that ERK is negatively regulated by an interplay of different phosphatases in a cell-context-dependent manner (Rintelen, 2003}.
The starting point of this study was the demonstration that mammalian DSPs not only function in Drosophila, but also exhibit strict specificities even when overexpressed. Considering the relatively low conservation of phosphatases at the sequence level -- ~50%-60% similarity -- this is somewhat surprising. It is, however, observed that DMKP3 is more active in flies than the mammalian ERK phosphatases MKP3 and MKP4, which were rather weak. However, different strengths of transgenes may also reflect insertion effects indicating that statements other than qualitative ones are difficult to make (Rintelen, 2003).
Several lines of evidence indicate that DMKP3 is an ERK-specific phosphatase and that it cooperates with PTP-ER. (1) DMKP3 dephosphorylates ERK but not JNK in vitro. (2) Overexpression of DMKP3 produces phenotypes resembling those of ERK but not JNK loss-of-function mutations. (3) Epistasis experiments using Dmkp3 gain-of-function and loss-of-function alleles indicate that DMKP3 acts in the RAS/ERK pathway in the eye and the wing. (4) The synthetic lethality of PTP-ER-; Dmkp3- double mutants is rescued by reducing ERK levels by half (Rintelen, 2003).
This interaction is reminiscent of the yeast DSP Yvh1 and the tyrosine phosphatase Ptp2, which have little effect when mutated alone, but double mutants are sporulation defective. Since there are five additional MKPs in the Drosophila genome, negative regulation of ERK by a combinatorial network of those phosphatases will probably reveal high redundancy as well (Rintelen, 2003).
In Dmkp3 mutant eyes, both R3 and R4 cells are misspecified in a small fraction of ommatidia. DMKP3 has an autonomous and a non-autonomous role in specifying R3 and R4. The autonomous DMKP3 function derives from the high, albeit not complete correlation of a Dmkp3- phenotype and a Dmkp3- genotype in the R3 and R4 cells. Because R3 and R4 are the most distantly related cells in the precluster, the high incidence of both R3 and R4 being mutant indicates a strong requirement for DMKP3 function in these cells. The evidence for a non-autonomous function of DMKP3 comes from phenotypically mutant ommatidia in which at least one cell of the R3/R4 pair is wild-type and from phenotypically mutant and genotypically wild-type ommatidia close to Dmkp3- clones (Rintelen, 2003).
Non-autonomous effects on outer photoreceptors were also observed for groucho, argos, fat facets, liquid facets, sidekick and atrophin clones. The results have been interpreted to indicate that surrounding cells participate in photoreceptor differentiation. The data presented here provide the first direct evidence that levels of RAS/ERK activity in cells surrounding the growing ommatidial cluster can influence ommatidial patterning. They may also explain why a Ras1 gain-of-function allele dominantly enhances the fat facets (faf) loss-of-function phenotype, although faf function resides outside the photoreceptors (Rintelen, 2003).
From these results it is inferred that the misdifferentiation of Dmkp3- ommatidia correlates with the behavior of the mystery cell. The mystery cell must leave the precluster to permit a physical interaction of R3 and R4 precursor cells to engage in a Notch-Delta-mediated specification of the R3 and R4 fate. In the absence of DMKP3 in R3 and R4 precursors and in the surrounding cell pool the mystery cell has a chance of being locked between R3 and R4, thus preventing the correct specification of its fate and that of the R3 and R4 precursors. The presence of misspecified R3/R4 cells without any intervening extra photoreceptor cells suggests that the mystery cell left the cluster too late and thus interfered with R3/R4 development (Rintelen, 2003).
How could cells surrounding the mystery cell be involved in eliciting its exit from the precluster? Conceivably, changes in cell adhesion, which may be regulated by an ERK signal, play a major role in expunging the mystery cells from the cluster. Upon recruitment of cells into the cluster, cell-cell contacts between photoreceptor cells are tightened. The mystery cells cannot adhere to the differentiating cells in the cluster and are expelled like melon seeds. As DMKP3 is not required in the mystery cells, it is probable that it is not the absolute value of cell-adhesive properties, but the relative amount compared with its neighbors that influences their behavior. This model implies that mutations altering cell-adhesive properties should lead to Dmkp3--like ommatidia. Indeed, loss of sidekick and atrophin, coding for adhesion molecules, result in a very similar phenotype by affecting cells outside the cluster. Furthermore, EGFR signaling and particularly ERK activity may not only influence cell fate, but also directly or indirectly influence cell adhesion. EGFR to ERK signaling has been shown to affect the adhesive properties of mammalian cells, and recent evidence in Drosophila also points to a role of EGFR in cell adhesion. High ERK activity has also been found in migrating cells, although activated ERK per se is insufficient to influence migration. The possibility to modulate RAS pathway activity in Drosophila almost at will may establish the developing eye as an interesting system in which the connection between RAS signaling and cell adhesion within an epithelium can be further analyzed (Rintelen, 2003).
In metazoan oocytes, a metaphase arrest coordinates the completion of meiosis with fertilization. Vertebrate mos maintains the metaphase II arrest of mature oocytes and prevents DNA replication between the meiotic divisions. A Drosophila homolog of mos has been identified and it was shown to be the mos ortholog by two criteria. The dmos transcripts are present in Drosophila oocytes but not embryos, and injection of dmos into Xenopus embryos blocks mitosis and elevates active MAPK levels. In Drosophila, MAPK is activated in oocytes, consistent with a role in meiosis. Deletions of dmos were generated; as in vertebrates, dmos is responsible for the majority of MAPK activation. Unexpectedly, the oocytes that do mature complete meiosis normally and produce fertilized embryos that develop, although there is a reduction in female fertility and loss of some oocytes by apoptosis. Therefore, Drosophila contains a mos ortholog that activates a MAPK cascade during oogenesis and is nonessential for meiosis. This could be because there are redundant pathways regulating meiosis, because residual, low levels of active MAPK are sufficient, or because active MAPK is dispensable for meiosis in Drosophila. These results highlight the complexity of meiotic regulation that evolved to ensure accurate control over the reproductive process (Ivanovskam, 2004).
Homology searches with the vertebrate MOS protein against the Drosophila genome identified the CG8767 open reading frame as its closest homolog. Xenopus MOS and CG8767 are 30% identical and 45% similar across the entire coding sequence. CG8767 is the only serine/threonine protein kinase in the Drosophila genome that was identified in the homology search when the BLASTP server was used and Xenopus MOS was used as a query; the kinase domain of CG8767 is more similar to Xenopus MOS than to any other protein kinase. Therefore, CG8767 is a good candidate for the Drosophila ortholog of MOS and will be referred to as Dmos (Ivanovskam, 2004).
The elevation of MAPK phosphorylation after injection of dmos in Xenopus embryos raises the possibility that the MAPK cascade is also active in Drosophila oocytes and that it may be downstream of Dmos. To test these hypotheses, a deletion of dmos was generated, and the level of MAPK and MEK1/2 (MAPK kinases) phosphorylation was analyzed in wild-type and dmos mutant flies. MAPK and MEK1/2 are phosphorylated in wild-type Drosophila ovaries, during prophase I (stages 1-13) and metaphase I (stage 14). Strikingly, the phosphorylation level of MAPK in the dmos mutant ovaries was reduced 15-fold in metaphase I-arrested oocytes and 4-fold in prophase I-arrested oocytes. The levels of MEK1/2 phosphorylation were also greatly reduced. In contrast, MAPK and MEK1/2 phosphorylation levels were unaffected in mutant female carcasses from which the ovaries had been completely removed and in mutant males, suggesting that Dmos does not affect the MAPK cascade outside the ovaries. These results indicate that the MAPK cascade is active in Drosophila ovaries and that Dmos is required for activation of MAPK specifically in the ovaries. A residual level of MAPK phosphorylation in the mutant ovaries suggests that MAPK can be phosphorylated by a redundant pathway (Ivanovskam, 2004).
Given the essential function of mos in vertebrates and the requirement for Dmos in activation of the MAPK cascade in Drosophila ovaries, the existence of dmos-redundant pathways was explored in three ways. (1) Another MAPKKK may activate MAPK in the dmos mutant ovaries, providing the residual 7% of activity and sufficient function to mask any phenotypic consequences of deleting dmos. The RAF-1 protein, a conserved homolog of the v-raf oncogene, activates the MAPK cascade in Drosophila somatic tissues and is therefore a good candidate for redundancy with Dmos. To test this hypothesis, one copy of a null raf-1 mutation was introduced in the dmos mutant background and thereby the levels of RAF-1 were reduced by half. The raf-1 mutation does not dominantly enhance the dmos phenotype, and thus either RAF-1 is not redundant with Dmos or the levels of RAF-1 are not limiting in the ovaries (Ivanovskam, 2004).
(2) The MAPK cascade activates the Cyclin B/CDK1 complex, so a second possibility is that Cyclin B/CDK1 is activated by a pathway redundant with Mos. In vertebrates, both mos and cyclin B are translationally activated by the CPEB protein. The Drosophila ortholog of CPEB is the oo18 RNA binding (Orb) protein. Analogously to CPEB, Orb may also function to translationally regulate mos and cyclin B. A role for Orb in establishment of the metaphase I arrest was explored and orbmel mutant ovaries were found to have normal both se I spindles in stage 14 oocytes. If cyclin B and mos are both targets of Orb, and if they function redundantly in establishing the metaphase I arrest, then reducing the amount of mos in a weak orbmel mutant may unmask a role for Orb in metaphase I arrest. However, it was found that reducing the levels of mos in the orbmel mutant background did not enhance the orbmel mutant phenotype, suggesting that Orb does not regulate a pathway parallel to MOS (Ivanovskam, 2004).
(3) The Pan Gu (Png) protein kinase complex is required for sustaining high levels of Cyclin B by activating cyclin B posttranscriptionally. Png and its two activating subunits, Plu and Gnu, promote mitosis specifically in the early embryonic divisions, but all of the proteins are present during oogenesis. Thus, Dmos and Png could act redundantly to control Cyclin B and active Cdk1/Cyclin B (or Cyclin B3) during meiosis. This hypothesis was tested by examining the phenotypes of double mutants, but no genetic interactions were observed between png and dmos. The stage 14 oocytes in the ovaries of png;dmos females had normal metaphase I spindles, and the embryos had the same phenotype as the png single mutant. Therefore, the Png pathway is not redundant with Dmos (Ivanovskam, 2004).
Additional mechanisms independent of MAPK may control female meiosis in parallel with dmos, as supported by observations of pathways acting in parallel to mos in vertebrates. For example, the APC inhibitor, Emi1, and the CyclinE/CDK2 complex have been shown to have cytostatic factor activity . The existing alleles of CyclinE and rca1, the Drosophila emi1 homolog, are lethal or disrupt the early stages of meiosis; thus, it has not been possible to test their role specifically in the metaphase I arrest (Ivanovskam, 2004).
This analysis of dmos illustrates the divergence of meiotic regulatory mechanisms and supports the emerging paradigm that meiosis is subjected to parallel, compensatory controls to ensure the proper completion of this developmental process that is critical for reproductive success. It will be important to evaluate the role of MAPK in Drosophila female meiosis as well as to test the requirements for CDK1/Cyclin activity, APC-mediated proteolysis, and the spindle checkpoint. The mechanism by which the metaphase I arrest is maintained and released is particularly intriguing. It has been demonstrated that chiasmata are essential for signaling the arrest, but the role of CDK1/Cyclin remains unknown. To date, a genetic evaluation of these components in oocytes has been hampered by the lack of alleles that could distinguish between roles during earlier mitotic divisions in the germline and later meiosis in the oocyte. Alternative approaches to producing conditional phenotypes are being developed, and these should permit such analyses in the future (Ivanovskam, 2004).
The mammalian receptor protein tyrosine kinase (RTK), Anaplastic Lymphoma Kinase (ALK), was first described as the product of the t(2;5) chromosomal translocation found in non-Hodgkin's lymphoma. While the mechanism of ALK activation in non-Hodgkin's lymphoma has been examined, to date, no in vivo role for this orphan insulin receptor family RTK has been described. This study describes here a novel Drosophila RTK, Alk, which maps to band 53 on the right arm of the second chromosome. Full-length Alk cDNA encodes a phosphoprotein of 200 kDa, which shares homology not only with mammalian ALK but also with the orphan RTK LTK. Analysis of both mammalian and Drosophila ALK reveals that members of the ALK family of RTKs contain a newly identified MAM domain within their extracellular domains. Like its mammalian counterpart, Alk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of Alk in the Drosophila brain, careful analysis reveals an additional early role for Alk in the developing visceral mesoderm where its expression is coincident with activated ERK (Lorén, 2001).
These data provide evidence for the existence of a Alk RTK pathway in Drosophila and show that ERK participates in this pathway, and that it is activated by Alk in vivo. Expression patterns of Alk, together with activated ERK, suggest that Alk fulfils the criteria of the missing RTK pathway, leading to ERK activation in the developing visceral mesoderm (Lorén, 2001).
Mammalian Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases (RTKs) which acquire their transforming capability when they are truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma. To date, several chromosomal rearrangements leading to an activated ALK RTK have been described, including NPM-ALK which are constitutively dimerized through the fused domain. However, there are few insights into the normal structure and function of the ALK RTK. Full-length cDNA encoding the mammalian ALK RTK has been identified as a first step towards a functional assessment of the receptor. ALK is a member of the Insulin Receptor superfamily, most closely related to the orphan RTK leucocyte tyrosine kinase (LTK). In situ hybridization studies have revealed ALK expression in the developing nervous system and ALK is currently a novel orphan receptor tyrosine kinase that is suspected to play an important role in the normal development and function of the nervous system (Lorén, 2001).
Alk was identified using a degenerate PCR approach. Alk is a 200 kDa RTK that has strong homology with both ALK and LTK. Due to the conserved nature of many receptor signalling systems in Drosophila, ALK RTK mediated signalling may also be conserved from Drosophila to vertebrates. Drosophila has a smaller number of RTK genes than vertebrates, with ~21 RTKs now predicted to be encoded by the Drosophila melanogaster genome. In addition, since the sequencing of the Drosophila melanogaster genome has now been completed it can now be said that while an Insulin Receptor homologue is present, there appears to be no homologue for the ALK relative RTK, LTK in Drosophila melanogaster. Alk is expressed during early mesodermal development as well as within the developing nervous system. Interestingly, early expression of Alk in the mesoderm correlates with ERK activation in the developing embryo mesoderm in vivo. Furthermore, using the UAS-GAL4 expression system, together with clonal over-expression techniques, Alk is observed to indeed activate ERK in vivo (Lorén, 2001).
To identify novel PTKs in Drosophila melanogaster, a degenerate PCR-based approach was used. Highly conserved residues within subdomains VIb and IX of known PTKs were targeted for degenerate PCR primer design, leading to the identification of several novel putative Drosophila melanogaster PTKs. Multiple PCR products were obtained and sequenced, identifying novel as well as previously described PTKs. One of the novel PCR products, displayed the greatest similarity to members of the mammalian Insulin Receptor RTK superfamily (Lorén, 2001).
To characterize the Alk protein, pcDNA3:Alk was transiently expressed in 293 cells. Anti-Alk antibodies were used to detect Alk from cell lysates. Lysates were resolved on SDS-PAGE and analysed by immunoblotting for Alk. Alk antibodies specifically recognized a 200 kDa protein, which is present when the cells were transfected with pcDNA3:Alk. Lysates were also analysed by anti-phosphotyrosine immunoblotting; Alk was detected as a 200 kDa tyrosine phosphorylated protein, suggesting that Alk is indeed a PTK. Furthermore, anti-Alk antibodies recognize a doublet of endogenous Alk at approximately 200 kDa from whole embryo extracts. Currently, the nature of this doublet is unknown; it may reflect the phosphorylation status of Alk, although alternative splicing may also be responsible (Lorén, 2001).
A 1997 study conducted by Gabay (1996) produced a detailed 'atlas of MAPK activation' in vivo. This study used antibodies that were specific for activated phospho-ERK as a tool for dissecting ERK activation throughout Drosophila embryonic development. It was noted that most aspects of the phospho-ERK pattern observed could be accounted for by known Drosophila RTK pathways. However, several of the patterns revealed were novel with respect to the receptor they are triggered by. It was speculated that these patterns may be induced by unknown RTKs that may activate ERK. In particular, prominent phospho-ERK staining was observed in the visceral mesoderm at stage 11. It was first seen as segmental patches, before fusion of the visceral arches from each segment, and subsequently observed as a continuous waved line. Furthermore, this phospho-ERK pattern in the visceral mesoderm was not dependent upon the Heartless RPTK. Since Alk expression was seen in the visceral mesoderm, whether Alk expression coincided with the phospho-ERK pattern in the visceral mesoderm was examined in vivo (Lorén, 2001).
In order to confirm that Alk and phospho-ERK were expressed in the visceral mesoderm during development, wild-type embryos were collected and stained for Alk and phospho-ERK. In both cases, expression was observed in the visceral mesoderm at stages 11/12 in a similar pattern. Subsequently, embryos were collected and double-stained for activated phospho-ERK and Alk. Co-localization of both activated phoshpo-ERK and Alk could clearly be observed in the visceral mesoderm (Lorén, 2001).
So far it has not been possible to obtain Alk mutants and so it was not possible to examine whether Alk is responsible for ERK activation in the developing visceral mesoderm in vivo. However, it was ask if Alk was capable of driving ERK activation in vivo by utilizing the GAL4-UAS system. Alk cDNA was cloned into P element expression vectors under the control of yeast GAL4 upstream activating sequences (UAS) and P element-mediated germ-line transformation was used to generate UAS:Alk transgenic fly lines. When Alk was expressed ectopically under the control of the Actin5C promoter driving GAL4 (Actin5C-GAL4) the result was 100% embryonic lethality. In order to examine whether the Alk RTK is capable of driving ERK activation in vivo, pGMR-GAL4, which drives expression in all photoreceptor cells, was employed to express Alk in the developing eye disc. A very clear effect of Alk expression on ERK activation was observed: normally prominent ERK activation is seen within the morphogenetic furrow, with lower levels in the differentiated third instar eye disc. In contrast, high levels of ERK activation in vivo were observed when Alk was expressed. Further conformation of Alk driven ERK activation in vivo was achieved using a combination of the FLP-out system and the GAL4-UAS system. In this system, a fragment of DNA bracketed by FRT sites and containing transcription stop signals is inserted between the Actin5C promoter and GAL4. Heat shock induction of Flippase activity induces recombination in which the transcription stop segment is flipped out, thereby allowing the Actin5C promoter to drive the GAL4 expression. This system allows the creation of clones of cells expressing Alk, which are marked by GFP expression. The expression of Alk, as judged by immunostaining, and GFP were coincident, demonstrating that the system works for Alk as well as establishing the specificity of the anti-Alk antibodies. While endogenous Alk protein is expressed in the third instar brain during normal development, levels of Alk within over-expressing clones are clearly observed over endogenous levels. Alk over-expressing clones also display increased levels of phosphotyrosine, consistent with the over-expressed Alk being active and either directly or indirectly leading to protein phosphorylation in these clones. Furthermore, larger clones were observed to disrupt the normal tissue structure, leading to abnormal disc development. Animals carrying Alk over-expression clones did not survive to adulthood. Further analysis of Alk clones in discs isolated from third instar larva indicates that Alk leads to ERK activation in situ. Thus, Alk has the capacity to drive activation of ERK in vivo, and is therefore a prime candidate for the 'missing' RTK driving ERK activation within the developing visceral mesoderm in vivo (Lorén, 2001).
The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. The Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily.
Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. Drosophila Alk was identified using a degenerate PCR approach (Lorén, 2001). Like its mammalian counterpart, DAlk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of DAlk in the Drosophila brain, careful analysis reveals an additional early role for DAlk in the developing visceral mesoderm where its expression is coincident with activated ERK (Lorén, 2001). In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as optomotor-blind-related-gene-1 (org-1), a Drosophila homologue of mammalian TBX1, in these cells (Lee, 2003).
Localized activation of the Ras/mitogen-activated protein kinase (MAPK) cascade in the visceral mesoderm has been noted previously. In the somatic muscle lineage this pathway is required for founder cell specification. It was therefore hypothesized that Jeb signals through the Ras/MAPK cascade in the visceral mesoderm. Activated MAPK is indeed detected in the visceral mesoderm precursors that take up Jeb. The observed overlapping signals for diphospho-MAPK and org-1, as well as the exclusive staining patterns for diphospho-MAPK and sns, confirm that the MAPK pathway is activated in presumptive visceral muscle founders. Moreover, Jeb signalling is necessary and sufficient to activate the Ras/MAPK cascade in visceral mesoderm precursors. Immunostaining of jeb mutant embryos demonstrates absent diphospho-MAPK in the ventral visceral mesoderm cells that normally accumulate Jeb and become founders. As with founder cell markers, ectopic Jeb produces ectopic diphospho-MAPK, but only in the visceral mesoderm (Lee, 2003).
The expanded expression of org-1 upon mesodermal expression of activated versions of Drosophila Ras and human Raf implicates the Ras pathway in MAPK activation and founder cell specification in the visceral mesoderm. If Jeb signals through the Ras/MAPK pathway, then activation of this pathway should rescue jeb mutations. This prediction is true. As judged by expression of Fasciclin III, a marker of visceral mesoderm differentiation, expression of activated Ras can substantially rescue jeb mutant embryos (Lee, 2003).
Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants are viable, but exhibit developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppresses the ERK-dependent differentiation, which is further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulates ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, these studies reveal a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila (Kim, 2006).
Many negative regulators of the Ras/ERK pathway including various dual-specificity phosphatases are transcriptionally induced by activation of the Ras/ERK pathway to form a negative feedback loop. Since RSK acts as a negative regulator of the Ras/ERK pathway, it is possible to hypothesize that expression of RSK may be induced by Ras/ERK signaling activity. However, the results clearly showed that RSK is ubiquitously expressed in all developmental stages, while Ras/ERK signaling is activated in a specific region and at specific times. Furthermore, although the expression of pnt-P1 was highly induced by hyperactive Ras (RasV12) and silenced by dominant-negative Ras (RasN17), RSK gene expression was not altered by Ras at all, suggesting the Ras/ERK signaling pathway does not transcriptionally induce RSK (Kim, 2006).
Genetic and biochemical analyses using kinase-dead mutants of RSK suggested that the kinase activity of RSK is dispensable for its role during Drosophila eye and wing development. This is in stark contrast to previous assertions on RSK as an important kinase that controls many crucial downstream targets of the Ras/ERK pathway through phosphorylation in mammals. Supporting the results, there were no differences in the phosphorylation level of histone H3, a well-known target of RSK, between wild-type and RSK-null eye and wing discs. Therefore, it is believed that the substrate phosphorylation by RSK is largely unnecessary for its function in Drosophila. However, since some phenotypes including life span reduction, fertility reduction and growth retardation shown in RSK-null flies are not significantly rescued by expressing kinase-dead mutants of RSK by the da- or hs-Gal4 driver, the possibility that the kinase function plays a role in developmental processes other than eye and wing development cannot be entirely excluded. In addition, since only one RSK isoform exists in Drosophila, the physiological function of RSK shown in this study may not satisfactorily represent more specialized physiological roles of all the RSK isoforms (RSK1-4) in mammals (Kim, 2006).
Through a biochemical study using rat PC12 cell line, it has been claimed that RSK negatively regulates the Ras/ERK pathway by phosphorylating the Son of sevenless (Sos) Ras-GEF protein, an upstream activator of Ras. However, genetic analyses using Drosophila did not coincide with this result. Expression of RSK strongly suppressed the phenotypes of the constitutively active forms of Ras and Raf which are downstream signaling molecules of Sos, suggesting that RSK-mediated inhibition of the Ras/ERK pathway does not occur through Sos in Drosophila. Moreover, kinase-dead RSK also completely inhibited Ras/ERK-dependent signaling in a similar manner to wild-type RSK, which further undermined the possibility of phosphorylation-dependent inhibition of Sos by RSK in Drosophila eye development (Kim, 2006).
Since the phosphorylation of ERK was thought as a prerequisite for its nuclear entry, it was also determined whether RSK negatively regulates ERK phosphorylation by inducing gene expression of MAP kinase-specific phosphatases (MKP). Interestingly, although RSK dramatically inhibited the nuclear migration of ERK, it did not affect the status of ERK phosphorylation. Rather, direct protein–protein association between RSK and ERK is the essential mechanism to inhibit ERK signaling by RSK, since the mutant forms of RSK defective in binding ERK completely failed to rescue the phenotypes of RSK-null flies and since wild-type RSK failed to suppress the phenotypes of ERKSem (Kim, 2006).
Interestingly, recent reports have demonstrated that ERK enters the nucleus by diffusion in a temperature-dependent manner, which may explain the temperature-sensitive phenotypes of RSKD1 flies. This suggests that the binding partner of ERK is necessary for the tight regulation of the ERK nuclear localization. Since RSK is constitutively cytoplasmic even in the presence of upstream activators such as RasV12, it is very likely that RSK appropriately maintains ERK activity by restraining ERK in a cytoplasmic compartment, which would prevent ERK from activating its nuclear targets. Consistent with this argument, the nuclear entry of activated ERK is dramatically increased by the loss of RSK. Collectively, these studies demonstrate that RSK is a critical negative regulator of ERK in Drosophila by acting as a cytoplasmic anchor(Kim, 2006).
The initiation of gene expression in response to Drosophila receptor tyrosine
kinase signaling requires the nuclear import of the MAP kinase, Rolled. However,
the molecular details of Rolled translocation are largely unknown. In this
regard, D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin have been identified. DIM-7 exhibits a dynamic nuclear
localization pattern that overlaps the spatial and temporal profile of nuclear,
activated Rolled. Co-immunoprecipitation experiments show that DIM-7 associates
with phosphorylated Rolled in Drosophila S2 cells. Furthermore, moleskin
mutations enhance hypomorphic and suppress hypermorphic rolled mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated Rolled. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel also reduce the nuclear localization of activated Rolled. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated Rolled (Lorenzen, 2001).
Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. This study shows that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and this cytoplasmic hold is required for vein cell fate. At the same time, MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. A novel Ras pathway bifurcation is proposed in Drosophila and the results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK (Marenda, 2006).
Msk is a Drosophila homolog of importin 7 (encoded by the moleskin gene, msk), which is a MAPK nuclear import co-factor. Msk expression can facilitate the nuclear translocation of pMAPK in vivo. To observe the phenotypic consequences of continuous and long-term reduction of
MAPK cytoplasmic hold, MSK was expressed in the posterior compartment of wing
discs (en:GAL4; UAS:msk or en::msk). Since the GAL4/UAS system was used to overexpress Msk in these discs, MAPK-GAL4 (MG) cannot also be used to detect MAPK nuclear translocation. Therefore, to
visualize MAPK, HSV epitope-tagged MAPK (hs:M) was used and stained
for the epitope tag. In control wings, the tagged MAPK is expressed at low
levels, and is not specifically concentrated in any one compartment. However, when Msk is overexpressed in the posterior compartment, tagged MAPK expression is
visibly elevated. A
closer analysis of this epitope shows that it is in many cell nuclei both
where hold normally occurs (e.g. the wing margin), and also in areas
where hold does not occur, confirming that Msk overexpression
increases the rate of MAPK nuclear translocation in the wing (Marenda, 2006).
This posterior ectopic expression of Msk eliminates pMAPK antigen within
pro-vein and margin cells in the posterior domain of the wing pouch. Surprisingly,
posterior Msk expression disrupts the anteroposterior compartment boundary, as
determined by GFP marking. High levels of cell death can disrupt development, and cause cells to cease to respect compartment boundaries. Since high levels of
cell death are seen in Msk overexpression wings, it is suggested that this may
explain the disruption. However, even when cell death is blocked with p35, loss of pMAPK is still observed in the posterior wing pouch,
suggesting that cell death alone is not the cause of lost pMAPK in this
genotype (Marenda, 2006).
Taken together, these experiments suggest that ectopic Msk can increase
MAPK nuclear translocation and overcome cytoplasmic hold, and that some
nuclear enzyme, most likely a phosphatase, then rapidly eliminates the pMAPK antigen (Marenda, 2006).
In cultured CCL39 cells, MAPK cytoplasmic tethering inhibits the ability of
cells to enter S phase, suggesting that MAPK nuclear translocation is important for
cell cycle entry. In the developing Drosophila larval wing, elevated
Ras signaling similarly promotes G1/S progression, and
MAPK loss-of-function mutations suppress this progression. Taken
together, these data suggest that the G1/S transition in the developing larval
wing may require MAPK nuclear translocation. Since cell proliferation in the wing
is better understood in larval rather than pupal stages, the analyses were focused at this stage (Marenda, 2006).
In larval wing discs, margin cells are non-proliferative [the zone of
non-proliferating cells (ZNC)], and markers of S-phase (BrdU) and M-phase (phospho-histone H3 antigen, pH3) are reduced in this territory.
Similarly, MG-driven GFP is also reduced in margin territories, indicating
that it too may be a marker for proliferation. However, MG-driven GFP is not
in the same cells as either BrdU or pH3 (phospho-histone H3 antigen, a marker of M-phase). In the developing
eye, MG-driven GFP follows the transcription of MG with a delay of 4-6 hours. Thus,
the observed non-coincidence of GFP with either BrdU or pH3 in the developing
wing may simply be due to this time lag (Marenda, 2006).
To analyze the cell cycle more precisely, FACS was used to determine the
cell-cycle phase of those cells expressing MG-driven GFP, following a 1-hour
induction and 6 hour recovery time. Sorting was performed for GFP and then the DNA content profiles
of the two cell populations (GFP– control cells with little
or no MAPK nuclear translocation versus GFP+ cells where MAPK
nuclear translocation has occurred) was compared. The GFP+ cell population has a
slightly elevated fraction in G2 and M phase, mostly at the expense of the
pool in G1. Although
these results are consistent with a function of MAPK nuclear translocation in
triggering proliferation, it remains possible that MG-driven GFP is a
consequence, not a cause of proliferation. To test this, MAPK
nuclear translocation was increased using NMG (MG fused with a SV40 nuclear localization sequence), while simultaneously driving GFP reporter
expression (hs:NMG, UAS:GFP). NMG was induced for 1 hour, followed by
6 hours recovery, and a dramatic reduction was seen in the fraction of
GFP+ cells in G1, while greatly raising the fraction in S and G2/M, suggesting that
nuclear translocation of MAPK is sufficient to induce proliferation. These larvae were then allowed to recover for 24 hours, the fraction of GFP+
cells in G2/M rose, at the expense of the pool in G1 and S. This suggests that
MAPK nuclear translocation is sufficient to induce S-phase transition in wing
cells, and after the initial nuclear MAPK-induced transition to S-phase, cells
then progress normally through the division cycle (at least as far as G2) (Marenda, 2006).
However, it could be that upon induction of NMG, a block in G2/M occurs,
and this allows cells to build up in S phase. To rule this out, hs:MG and hs:NMG were expressed, and pH3 staining was analyzed. But no difference in pH3-positive nuclei was observed in hs:NMG discs versus
hs:MG controls. Indeed, more pH3-positive nuclei are seen in
hs:NMG wing pouches when compared with hs:MG controls, along
with increased pH3 staining in the ZNC. These data are
consistent with ectopic nuclear MAPK inducing cell proliferation, even in
populations of cells that are normally non-proliferative (Marenda, 2006).
Continuous posterior-compartment driven Msk expression
(en::msk) was used as a second test to determine the role of MAPK nuclear
translocation in wing cell proliferation. Again, the fraction of
GFP+, S-phase cells is increased (27% versus 16% for the control,
anterior compartment GFP- cells), as is the fraction in G2/M
(37% versus 32%), at the expense of cells in G1. Since Msk is
continuously available in this experiment, this is interpreted as a summation of
the transient 6 and 24 hour effects seen with NMG. Consistent with this, in
en::msk discs, elevated posterior compartment expression
of the S-phase limiting factor Cyclin E, the M-phase
limiting factor String (stg:lacZ) and the S-phase
marker BrdU, are seen. Taken together, these data suggest that MAPK nuclear
translocation does indeed normally promote S-phase transition in developing
wing cells (Marenda, 2006).
Elevated proliferation in the posterior compartment might be expected to
produce adult wings with enlarged posteriors (the 'J.Lo wing'). However,
prolonged and elevated expression of Msk induces caspase-dependent cell death
and the resulting
adult wings are severely disrupted, with nearly normal anterior compartments
and severely reduced posteriors (the 'Twiggy wing'). These wings display loss
of posterior tissue, including distal regions of veins L4 and L5, and fused
posterior and anterior crossveins (Marenda, 2006).
Reduction of EGFR pathway function via loss of one copy of the gene
encoding MAPK (rl10A) strongly
suppresses the Msk-induced Twiggy wing, consistent with the Msk overexpression
phenotype being dependant on MAPK. If Msk is limiting in the
wing, then msk gene dose should affect vein
formation. msk gain-of-function should suppress vein formation, while
msk loss-of-function should enhance vein formation (Marenda, 2006).
To examine Msk gain-of-function, overexpression of the negative
ligand Argos in the posterior compartment of the wing, which leads to vein
loss 100% in vein L4, and 90% in vein L5, was examined. When
Msk and Argos are co-expressed, Msk enhances the vein loss phenotype of Argos
to 100% in L4 and 100% in L5. Similarly,
overexpression of the nuclear ETS domain transcription factor Pointed P2
(PntP2, a positive MAPK effector) induces vein loss in 0% in L4 and 89% in L5,
consistent with the suggestion that MAPK nuclear function antagonizes vein
fate. Co-expression of Msk and PntP2 further enhances this vein loss to 97% in
L4 and 100% in L5 (Marenda, 2006).
To examine msk loss of function, interactions were examined of a
msk null allele (msk5) with a rho
gain-of-function allele (hs-rho30a, and a rolled
gain-of-function allele (rlSem), both of which dominantly
cause extra vein formation. Trans-heterozygous
hs-rho30a/msk5 wings show a strong enhancement
of the rho extra-vein phenotype. Similarly,
trans-heterozygous rlSem/msk5 wings also show
enhancement of the rolled extra vein phenotype. Furthermore,
Msk gain of function suppresses the extra veins caused by both
hs-rho30a and UAS:rlSem expression (Marenda, 2006).
Though these effects may reflect additive genetic phenotypes as opposed to
true genetic interactions, when taken together, gain-of-function and
loss-of-function data suggest that Msk normally functions to restrict vein
formation. It is suggested that this is because gain of msk function leads to
increased nuclear MAPK (vein loss), while loss of msk leads to
increased cytoplasmic MAPK (extra veins). These data are consistent with the
suggestion that vein formation through MAPK occurs through a
cytoplasmic, rather than a nuclear target (Marenda, 2006).
In summary this study has report the existence and contribution of MAPK cytoplasmic hold in the developing Drosophila wing. A difference was observed in cytoplasmic versus nuclear function of MAPK, and it is suggested that in the developing wing, MAPK subcellular localization controls the difference between vein specification (cytoplasmic MAPK) and proliferation (nuclear MAPK).
Perhaps vein differentiation is simply an indirect effect of repressing
cell proliferation by inhibiting MAPK nuclear translocation. To address this,
vein formation was analyzed in adult wings overexpressing both positive and
negative cell cycle regulators. If
vein formation is lost by inducing cell proliferation with effects other than
forced nuclear MAPK, this would argue that the observed vein loss in
en::msk is most probably due to an indirect effect of disrupting cell
proliferation, as opposed to disrupting cytoplasmic pMAPK (Marenda, 2006).
Overexpression of either CycE or Stg leads to increased proliferation in
Drosophila wings; however, there is little to no effect on vein formation,
with no vein loss in either case. Similarly, inhibiting cell proliferation by over-expressing either the cyclin-dependant kinase inhibitor dacapo, or the S-phase inhibitor p21 had no significant effect on vein formation. This is consistent with a direct effect for MAPK cytoplasmic hold on vein differentiation (Marenda, 2006).
There are a number of known cytoplasmic targets of MAPK, including p90RSK,
cPLA2 and Myosin light chain kinase (Ebisuya, 2005).
However, it is important to consider that some cytoplasmic target proteins for
MAPK may first be phosphorylated in the cytoplasm and then translocate to the
nucleus, or be inhibited from doing so, such as SV40 T-antigen and
Xenopus nucleoplasmin. In fact, it has recently been reported that the
co-repressor Groucho is directly phosphorylated by MAPK, and this
phosphorylation weakens its repressor activity, leading to extra veins
(Hasson, 2005). Groucho, though it functions as a nuclear transcription factor, may be phosphorylated in the cytoplasm in pro-vein cells, where it can then
translocate to the nucleus to affect changes in Notch transcription, leading
to vein formation (Marenda, 2006).
Recent reports suggest that MAPK cytoplasmic hold may perform similar
functions in mammals (Ebisuya, 2005). In vertebrate cells, expression of the death effector PEA-15 can sequester pMAPK in the cytoplasm.
After treatment with Retinoic acid, embryonic stem and carcinoma cells stop
proliferating, restrict the nuclear entry of pMAPK and differentiate into
primitive endoderm (Smith, 2004). In the mouse embryo, pMAPK is detected in the cytoplasm rather than the nuclei of cells receiving FGF signals
(Corson, 2003). A family of proteins called SEFs antagonize MAPK signaling
(Fürthauer, 2002). More recently, SEF has been found to act directly to hold pMAPK in the cytoplasm, suggesting a mechanism for FGF pathway attenuation through MAPK cytoplasmic hold. No homolog of PEA-15 or
SEF has been identified outside the chordates by conventional bioinformatic techniques. However, a
fly protein with a function that is very similar to SEF would fit the MAPK
cytoplasmic hold phenomena observed in the eye and wing (Marenda, 2006).
While anchoring of pMAPK has been shown to restrict MAPK nuclear entry in
cell culture, it remains possible that pMAPK nuclear import could be prevented
by removing a required nuclear import co-factor. Thus, by cytoplasmic
sequestration of Msk (for example), pMAPK would be unable to translocate into
the nucleus, and pMAPK cytoplasmic hold would be achieved (Marenda, 2006).
Mammalian importin 7 is reported to import several proteins into the
nucleus, including histone H1, core histones, HIV-1 reverse transcription
complexes and the glucocorticoid receptor.
However, the current data suggest that MAPK is a crucial target for the phenotypes
observed in wings overexpressing Msk: (1) a null mutation in
Drosophila MAPK strongly suppresses the en::msk adult wing
phenotype; (2) increased nuclear MAPK is observed after overexpression of Msk
in larval wings; (3) loss-of-function mutations in Drosophila Histone
H1 [Su(var)205] have no effect on the en::msk phenotype; (4) loss-of-function mutations in members of other vein
promoting pathways (thick veins, tkv8) have no effect on
the en::msk adult wing (Marenda, 2006).
In the developing compound eye, breaking MAPK cytoplasmic hold in cells
within the morphogenetic furrow results in reduced expression of Atonal, which
is required for the initiation of differentiation in the developing eye. Taken
together with new data from the developing wing, it is suggested that MAPK
cytoplasmic hold may be generally required for the cell cycle arrest necessary
for the initiation of differentiation, thus defining a novel bifurcation in
the Ras pathway to control different cellular outcomes. Finally, the
regulation of MAPK cytoplasmic hold may help to distinguish the MAPK signals
for cell fate from those for cell proliferation (Marenda, 2006).
The Drosophila Mitogen Activated Protein Kinase (MAPK) Rolled is a
key regulator of developmental signaling, relaying information from the
cytoplasm into the nucleus. Cytoplasmic MEK phosphorylates MAPK (pMAPK), which
then dimerizes and translocates to the nucleus where it regulates
transcription factors. In cell culture, MAPK nuclear translocation directly
follows phosphorylation, but in developing tissues pMAPK can be held in the
cytoplasm for extended periods (hours). This study shows that Moleskin antigen
(Drosophila Importin 7/Msk), a MAPK transport factor, is sequestered
apically at a time when lateral inhibition is required for patterning in the
developing eye. It is suggested that this apical restriction of Msk limits MAPK
nuclear translocation and blocks Ras pathway nuclear signaling. Ectopic
expression of Msk overcomes this block and disrupts patterning. Additionally,
the MAPK cytoplasmic hold is genetically dependent on the presence of
Decapentaplegic (Dpp) and Hedgehog receptors (Vrailas, 2006).
Early in eye development, all cells anterior to the furrow (phase 0) are
primed for Ras-induced neural differentiation; ectopic activation of the
pathway causes all cells to differentiate as photoreceptors, even without
atonal. Normally these cells are thought to receive only low levels
of Egfr-mediated Ras signaling, supporting proliferation but not
differentiation. Later, in the furrow (phase 1), Delta-induced,
Notch-mediated lateral inhibition progressively restricts Atonal expression to
single founder cells. Suspension of Ras signaling is required for this inhibition
in order to avoid premature neuronal differentiation, and it has been proposed
that this inhibition is mediated by MAPK cytoplasmic hold. However,
this block to the Ras pathway must be released in phase 2 (posterior to the
furrow) to allow for developmental induction by the R8 cell. To better
understand how MAPK cytoplasmic hold is maintained in phase 1, the
role was examined of the pMAPK nuclear transport factor Drosophila Importin 7/Msk, in eye development (Vrailas, 2006).
It is suggested that in wild-type eye discs, the level of pMAPK antigen is a
very misleading reporter of Egfr/Ras pathway activity, because cytoplasmic
hold in phase 1 allows even a relatively low level of pathway activity to
build up high levels of pMAPK antigen. A system has been developed to
reveal MAPK nuclear translocation without the use of an antibody (MG-driven
reporter gene expression that reveals MAPK nuclear translocation). [Note:
MG (Mapk-Gal4vp16) contains the entire sequence of Rolled, followed by the yeast GAL4 DNA binding domain (which is not known to contain a nuclear localization signal) with an acidic activation domain from herpes simplex virus protein 16].
However, it has been since found that under all conditions tested, MG-driven reporter expression does not reveal nuclear MAPK in phase 0,
where Ras pathway activation is required. MG-driven reporter
expression is reliably see in phase 2, where there is thought to be high (or sustained) levels
of Ras pathway activity. In phase 1, the level of pathway signaling may be
insufficient for expression, and thus MG-driven reporter expression may reveal
only high (or sustained) levels of nuclear MAPK. Alternatively, this could be
caused by a technical limitation: the hsp70 promoter drives the
expression of only low levels of MG protein.
Therefore, two less direct assays were used, that together, are
interpreted as revealing the loss of MAPK cytoplasmic hold in the furrow: (1)
loss of Atonal expression (as previously demonstrated by fusing an SV40 NLS to
MAPK and by the ectopic expression of Rasv12); and (2)
loss of pMAPK antigen, which may be due to exposure to a nuclear
phosphatase/protease (Vrailas, 2006).
The MAPK nuclear transport factor Drosophila Importin
7/Msk is apically sequestered in phase 1, the time when pMAPK nuclear access
is blocked. Furthermore, ectopic Msk is sufficient to break the
cytoplasmic hold in the furrow, as seen by loss of pMAPK antigen and
suppression of the early stages of Atonal expression. However, this transient
expression of Msk is unable to promote the precocious neural differentiation
or the increase in rough expression, as has been seen with
hs:rasv12 or nuclear-directed MAPK. Because ectopic
rasv12 produces an increase in pMAPK, and the
phosphorylation state of nuclear-directed MAPK is not required for nuclear
translocation, it may be that the available pool of pMAPK that can be imported
into the nucleus by Msk is enough to affect Atonal expression, but not to
affect Elav or Rough expression. Genetic evidence shows that the MAPK
cytoplasmic hold depends on the Hedgehog receptor Smo and is enhanced by the
loss of the Dpp receptor Tkv. smo loss-of-function clones reduce
Atonal and pMAPK expression, whereas tkv clones have much weaker
effects. However, the loss of smo and tkv together completely abolishes both pMAPK and Atonal expression in the furrow. This is consistent with a previous report of the loss of Atonal expression in smo tkv clones. Additionally, MAPK cytoplasmic hold in smo tkv clones is rescued by the additional loss of msk. Thus, msk genetically antagonizes pMAPK levels in the morphogenetic furrow: msk gain-of-function reduces pMAPK and msk loss-of-function (in smo tkv clones) increases it (Vrailas, 2006).
Hedgehog signaling has also been reported as a positive regulator of Atonal
on the anterior side of the furrow and as a negative regulator (perhaps
through Rough or Bar) on the posterior side. However,
the inductive effect of Hedgehog on Atonal appears to be independent of the
Hedgehog pathway transcription factor Ci, which is consistent with an indirect effect through the MAPK cytoplasmic hold. smo tkv msk triple mutant clones were used to show that msk is genetically epistatic to smo and tkv in the furrow, and suggest that Msk sequestration in the furrow is required for MAPK cytoplasmic hold, and that smo and tkv are genetically upstream of this sequestration of Msk. Indeed, loss of smo and tkv results in a disruption of the actin cytoskeleton in the furrow, as well as of expression of Egfr and other signaling molecules. The loss of apical constriction may therefore disrupt Msk apical sequestration in such a way as to allow precocious Msk-mediated pMAPK nuclear import (Vrailas, 2006).
What is more surprising is that differentiation and ommatidial assembly,
which are known to require Ras signaling and MAPK
nuclear translocation, occur normally in the absence of Msk in phase 2. It may be
that cytoplasmic MAPK targets are important for ommatidial assembly or that
pMAPK can translocate into the nucleus by some Ran-independent mechanism.
However, the possibility is favored that, in phase 2, other (possibly redundant)
transport factors are expressed (Vrailas, 2006).
Like the Ras pathway, msk plays a role in ommatidial rotation but
not chirality. It may be that in the absence of Msk, enough pMAPK can
translocate into the nucleus for ommatidial assembly, but not enough for
proper rotation. Additionally, in phase 0, Msk is found to be required for
proliferation, which also requires Ras signaling. Therefore, Msk is required
for some pMAPK nuclear translocation in phase 0 and phase 2, but is not
necessary in phase 1, in order to allow for the initial specification of the
Atonal-positive R8 (Vrailas, 2006).
To conclude, the apical sequestration of
Drosophila Importin 7/Msk in the morphogenetic furrow has been identified and it is suggested
that this may be required for the MAPK cytoplasmic hold in the developing eye.
Cytoplasmic hold is required to allow initial patterning through lateral
inhibition and the focusing of the proneural factor Atonal. It is further suggested
that this is mediated by the combined action of Hedgehog and Dpp (Vrailas, 2006).
FGF signalling is needed for the proper establishment of the mesodermal cell layer in Drosophila embryos. The activation of the FGF receptor Heartless triggers the di-phosphorylation of MAPK in the mesoderm, which accumulates in a graded fashion with the highest levels seen at the dorsal edge of the mesoderm. This study examines the specific requirement for FGF signalling in the spreading process. Only the initial step of spreading, specifically the establishment of contact between the ectoderm and the mesoderm, depends upon FGF signalling, and unlike the role of FGF signalling in the differentiation of heart precursors this function cannot be replaced by other receptor tyrosine kinases. The initiation of mesoderm spreading requires the FGF receptor to possess a functional kinase domain, but does not depend upon the activation of MAPK. Thus, the dispersal of the mesoderm at early stages is regulated by pathways downstream of the FGF receptor that are independent of the MAPK cascade. Furthermore, the activation of MAPK by Heartless needs additional cues from the ectoderm. It is proposed that FGF signalling is required during the initial stages of mesoderm spreading to promote the efficient interaction of the mesoderm with the ectoderm rather than having a long-range chemotactic function, and this is discussed in relation to the cellular mechanism of mesoderm spreading (Wilson, 2005).
Morphogenesis of the mesodermal cell layer has been considered to depend
entirely on FGF signalling, but in fact, FGF signalling is essential only for
the initial establishment of contact between mesoderm and ectoderm, and for
the late heart-differentiation signal, and these two processes are independent
and experimentally separable. Dominant-negative FGF-receptor constructs
disrupt differentiation, but do not affect spreading when expressed after the
initial contact has been made. Conversely, constitutively active tyrosine kinases other than FGF receptors expressed in htl mutants rescue late differentiation, but not early spreading. Similarly, in the mutants of the RhoGEF pbl, no early contact is made, and spreading is therefore inefficient, but cells that reach the dorsal region of the mesoderm are able to respond to FGF, activate MAPK, and differentiate into heart precursors (Wilson, 2005).
As the mesoderm spreads out over the surface of the ectoderm, the mesodermal cells that are in contact with the ectoderm accumulate high levels of the active form of MAPK. The fact that this accumulation of active MAPK is seen only in embryos with a functional FGF-signalling system in the mesoderm, but not in htl or dof (stumps or heartbroken) mutant embryos, indicates that it is triggered by the FGF receptor. Htl and Dof are expressed throughout the mesoderm, which suggests that the local activation of MAPK is induced by the local availability of a ligand, consistent with the expression pattern of the recently discovered ligands for Htl in the ectoderm. However, even a constitutively active form of Heartless expressed throughout the mesoderm, which is able to rescue spreading in htl mutants, only mediates MAPK activation at early stages in the cells directly apposed to the ectoderm. It is concluded that the presence of an activated form of the FGF receptor is not sufficient to trigger MAPK activation in mesodermal cells (Wilson, 2005).
This result may appear to contradict earlier studies showing the ability of activated FGF-receptors to trigger MAPK activation throughout the mesoderm, but the embryos in these studies were not analysed during the phase of the earliest contact of the mesoderm with the ectoderm, but rather at later stages, just before the time when MAPK activation normally occurs in the heart precursors in the dorsal region of the mesoderm. This phase of FGF-dependent MAPK activation in the mesoderm clearly has different requirements from the early phase, as is also shown by the results using other RTKs or downstream effectors of the RTK signalling pathway. These experiments demonstrate that signals from activated Raf cannot be transduced to MAPK in the cells during the early phase, except in the presence of an activated FGF receptor. It is concluded that, in addition to the signal from an activated RTK via Raf, a second event is necessary for MAPK to become phosphorylated. This event could either generate a second positive signal, or it could lead to the release of a negative, inhibitory signal (Wilson, 2005).
Two points suggest that the event depends on contact of the mesodermal cells with the ectoderm: (1) Lambda-htl (receptor that dimerizes spontaneously and becomes autophosphorylated in a ligand-independent fashion) induces MAPK phosphorylation only in mesodermal cells contacting the ectoderm, although it is expressed at uniform levels in all mesodermal cells; (2) the phenotype of pbl mutants supports this view. As in htl and dof mutants, the early contact of the mesoderm with the ectoderm fails to be made in pbl mutants, and mesoderm spreading is impaired. At later stages, Htl is able to trigger MAPK phosphorylation in the dorsal part of the mesoderm of pbl mutants, showing that FGF signalling in the mesoderm as such does not depend on pbl. By contrast, the early activation of MAPK is abolished. It is therefore argued that contact is a prerequisite for early FGF-receptor induced MAPK activation (Wilson, 2005).
Both the establishment of mesoderm-ectodermal cell contact and the activation of MAPK require the kinase domain of the FGF receptor to be intact, which suggests that these events depend upon a substrate of the FGF receptors not recognised by other activated receptor tyrosine kinases. One possibility is that this substrate is Dof, which is specifically phosphorylated by an activated FGF receptor. In this situation, Dof would provide a unique function that cannot be substituted by other activated receptor tyrosine kinases. Alternatively, this substrate could be a second receptor that is activated upon contact of the mesoderm with the ectoderm, or a component that acts in, or on, a pathway triggered by the engagement of the mesoderm with the ectoderm (Wilson, 2005).
It seemed likely that the early morphogenetic activity might require changes in subcellular architecture involving cytoskeletal regulators. Indeed, the establishment of contact between the mesoderm and the ectoderm is affected by mutations in the gene encoding the RhoGEF Pebble, and, as shown in this study, a reduction in the level of Rho and Rac proteins within the embryo. It is not known whether the Rho-family GTPases act downstream of or in parallel with FGF signalling. The defects of htl mutants cannot be rescued by the expression of an activated form of Rac or Cdc42. Thus, if Rac acts downstream of the FGF receptor, it is not in a simple epistatic pathway but requires the activation of other pathways as well. Alternatively, FGF signalling may act in conjunction with a separate pathway that directs the activity of the Rac proteins to promote contact between the mesoderm and the ectoderm (Wilson, 2005).
Spreading of the mesoderm on the ectoderm leads to a redistribution of mesodermal cells away from the site of invagination towards the dorsal edge of the ectoderm. This is often considered to be a process of directed cell migration. In this view, the graded distribution of activated MAPK levels in the nuclei of the mesodermal cells is suggestive of a response to a chemotactic signal originating from the target region. Both the expression pattern of the Htl ligands and the phenotypes of mutants in which the fate of the target region has been changed are inconsistent with this view. The activation of Heartless appears to be permissive for mesoderm spreading and it is suggested that FGF signalling functions primarily to promote the efficient interaction of the entire mesodermal primordium with the surface of the ectoderm and that this could act to impose order during the transition from an epithelial to a mesenchymal state. Simple spatial constraints could lead to an apparently directed migration. With the mass of mesodermal cells initially concentrated near the site of invagination, the only direction available for migration is away from this site. Hence, a signal-inducing motility would automatically promote directional movement. The dispersal of the mesoderm mass in dof mutants is noticeably improved by blocking cell division, and it is believed that this might be due to the smaller number of cells in the mesodermal primordium having greater access to the surface of the ectoderm (Wilson, 2005).
These observations raise the questions of how mesodermal cells spread over the surface of the ectoderm, and how activated MAPK accumulates in a graded fashion. A number of possibilities can be envisioned to account for the migration of the mesoderm. For example, the first cell that makes contact with the ectoderm could crawl over the ectoderm and function as the 'leading' cell of the mesodermal sheet. The other cells of the mesoderm tube would make contact with the ectoderm sequentially to follow the leading cell as it migrates dorsally. In this case, the MAPK gradient would be explained by the accumulation of the highest levels of activated MAPK in the cells that had been in contact with the ectoderm for the longest period of time. Alternatively, the cell that makes the initial contact with the ectoderm could remain largely stationary, and other mesodermal cells would reach the ectoderm by crawling over that cell. Once a mesodermal cell is in contact with the ectoderm, motility of the cell would cease, as in the process of 'boundary capture' described for mesodermal cells in Xenopus reaching the notochord during convergence movements. In this model, contact between the ectoderm and mesoderm would have an important role in establishing the single cell layer of mesoderm that covers the surface of the ectoderm at later stages. The MAPK gradient can be explained in this case by a transient activation of MAPK, which is downregulated once motility ceases, a model that is more consistent with known feedback mechanisms that operate during signal transduction. This model implies that the cells with the highest level of MAPK at the edge of the mesoderm would have only just come into contact with the ectoderm. In order to distinguish between the two mechanisms, cell labelling experiments will be required (Wilson, 2005).
FGF signalling is only one of many mechanisms that contribute to the establishment of the mesodermal cell layer. It is not essential for migration as such, but is clearly important for the orderly dispersal of mesodermal cells away from their site of invagination. These results suggest that FGF-signalling facilitates cell spreading by promoting the apposition of the invaginated mesodermal epithelium against the ectoderm (Wilson, 2005).
Continued: MAP kinase Protein Interactions part 2/3 | part 3/3
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