Regulation of Rolled Activity

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).

PTP-ER, a novel tyrosine phosphatase, functions downstream of Ras1 to downregulate MAP kinase during Drosophila eye development

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).

The Ste20 kinase Misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals

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).

A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK

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).

The Drosophila dual-specificity ERK phosphatase DMKP3 cooperates with the ERK tyrosine phosphatase PTP-ER

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).

Drosophila contains a mos ortholog that activates a MAPK cascade during oogenesis and is nonessential for meiosis

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).

Jelly belly protein activates the receptor tyrosine kinase Alk which acts through ERK to specify visceral muscle pioneers

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).

Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers

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).

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development

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).

Nuclear import of Rolled

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).

The activation of ERK represents the focal point of a conserved signaling module used by a diverse array of extracellular stimuli. The potency and duration of ERK activation and its accompanying translocation to the nucleus can profoundly affect the fate of a cell. This is apparent in PC12 cells where the decision to proliferate or differentiate depends upon the number and duration of receptors stimulated. Throughout development, cells respond to spatial and temporal signals and must interpret gradients to produce qualitative differences in gene expression. In Drosophila, the terminal system or Torso RTK signaling pathway illustrates one example whereby quantitative differences in D-ERK activity generate distinct cell fates. Distinct quantitative levels of D-ERK activity inside a cell may be achieved within the RTK pathway by modulating D-ERK phosphorylation. Moreover, it is apparent that mechanisms exist to control the localization of ERK activity by either regulating its retention in the cytoplasm and/or its nucleocytoplasmic shuttling. In this regard, nuclear translocation of dpERK is not always a compulsory consequence of RTK signaling. In Drosophila as photoreceptors are recruited into the developing retina, dpERK is held in the cytoplasm for up to several hours following EGFR and Sevenless RTK signaling. This raises the possibility that import is differentially controlled relative to D-ERK phosphorylation and/or dimerization. Interest in the active transport of dpERK also partly stems from the observation that dimer formation is a common property of mammalian MAP kinase family members (Lorenzen, 2001).

Presumably, D-ERK shares this property, since the residues involved in dimer formation have been conserved. Although monomeric dpERK can enter the nucleus passively, it has been shown that import of dimeric ERK is an active process. This study has clarified mechanistic issues in the nuclear relocalization of dpERK through the identification of DIM-7, the Drosophila homolog of importin 7 and member of the importin superfamily of nuclear transport proteins. DIM-7 exhibits several properties that establish its participation in RTK signaling. These include a physical interaction with CSW and dpERK, and the finding that DIM-7 is tyrosine phosphorylated in stimulated cells. Furthermore, alleles of msk, the gene that encodes DIM-7, dominantly interact with hypomorphic and hypermorphic alleles of D-ERK (Lorenzen, 2001).

The primary structure of DIM-7 originally suggested that it might play a role in nuclear transport. In addition to exhibiting significant sequence identity with its Xenopus and human homologs, DIM-7 also possesses a conserved Ran-binding domain. This latter property is a hallmark of nuclear transport proteins that display a RanGDP versus RanGTP regulated interaction with their cargo proteins. In addition to physically binding to phosphorylated D-ERK, DIM-7 and dpERK have overlapping nuclear localization patterns in developing tissues subject to RTK regulation. This made dpERK an attractive candidate cargo for DIM-7. To address this possibility during embryogenesis and in the absence of functional DIM-7, tracheal placodes and tracheal pits were assayed for defects in the accumulation of dpERK. Significantly, embryos in which the genomic interval encoding the msk gene is deleted or mutated exhibit a fivefold reduction in the number of dpERK-positive nuclei. Importantly and demonstrating that DIM-7 is essential for nuclear uptake of dpERK, expression of wild-type DIM-7 in a msk mutant background restores dpERK nuclear accumulation. These findings reinforce the idea that not only is dpERK actively transported through the nuclear pore complex but also that DIM-7 functions as either the transport receptor and/or adapter for dpERK (Lorenzen, 2001).

Finally, this study has implicated KET in the nuclear import of dpERK. As vertebrate importin 7 and importin b form an abundant heterodimeric complex, it was asked whether the Drosophila homolog of importin b, KET participates in the dpERK transport mechanism. Supporting a model whereby DIM-7 and KET function together in the nuclear transport of dpERK, it was found that nuclear localization of dpERK is impaired in homozygous ket mutant embryos. Although there are other possibilities, two models are envisioned by which DIM-7 could function in the nuclear import cycle of dpERK. In one, DIM-7 and KET could function together in the same import cycle, where DIM-7 and KET serve as the import adapter and import receptor, respectively. Alternatively, DIM-7 and KET could function independently of each other in separate import cycles that could be, at least partially, redundant. However, it is unlikely that DIM-7 and KET serve totally redundant functions for two reasons. First, each individual locus when deleted (msk) or mutated (msk and ket) reduces substantially the number of dpERK-positive nuclei, and, second, both msk and ket mutations, alone, are lethal (Lorenzen, 2001).

In the literature there appears to be an increasing number of transport receptors with complex cargo specificities. If importin 7 is the functional vertebrate homolog of DIM-7, then this transport factor can import at least three different proteins, dpERK, histone H1 and rpL23a. An additional point concerning specificity of DIM-7 regards its nearest homolog, Nmd5p, in Saccharomyces cerevisiae. Nmd5p is essential for the nuclear import of HOG1, a p38 MAP kinase family member that is activated in response to osmotic stress. Interestingly, the movement of HOG1 into the nucleus does not require the importin b homolog, RSL1. This work raises the possibility that DIM-7 might also mediate the nuclear transport of one or more other Drosophila MAPkinase family members, D-p38a (Mpk2), D-p38b (Mpk34C) and D-JNK (JUN kinase). The combinatorial use of different transport factors may provide a means for specific recognition of the various MAP kinase family members. For example, DIM-7 alone may bind and import D-p38; however, recognition of dpERK may require the simultaneous pairing of DIM-7 and KET. Determining the mechanism(s) employed to establish recognition between a cargo and its transport receptor will require a precise molecular dissection of the interactions between several transport receptor/cargo pairs (Lorenzen, 2001).

CSW was first demonstrated to have a positive function during embryogenesis in the Torso RTK pathway. In this pathway CSW serves two functions. First, the adapter protein DRK does not bind Torso; instead, CSW functions as an adapter linking Torso to RAS. Second, CSW is able to dephosphorylate the Torso autophosphorylation site that binds RasGAP. The work presented in this paper suggests a third function for CSW as an adapter to facilitate the physical interaction of DIM-7 with its import cargo dpERK. When two, or three, of these CSW functions are used within one signaling module, interpreting the epistatic relationships of CSW with various signaling components could become problematic. For example, previous epistasis experiments have suggested that CSW carries out its function either upstream or downstream of RAS1 and/or D-RAF. Now it appears these differences could simply reflect the differential use of the various signaling capabilities of CSW within a given RTK pathway (Lorenzen, 2001).

Whether or not the association of DIM-7 with CSW constitutes part of a regulatory process at the level of the receptor that governs D-ERK redistribution has yet to be determined. However, it appears that nuclear import is a fundamental mechanism used by cells to modulate incoming signals throughout development. It is expected, then, that the development of reagents to modulate the nuclear entry of specific molecules may have profound effects for controling both disease and oncogenic states (Lorenzen, 2001).

MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

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). Regardless of the mechanism, MAPK cytoplasmic hold may be a conserved mechanism necessary for the differentiation of certain developing tissues in many taxa, and proper control of MAPK subcellular localization may act as a developmental signal to determine the proliferative state of a cell (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).

smoothened and thickveins regulate Moleskin/Importin 7-mediated MAP kinase signaling in the developing Drosophila eye

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 and the mechanism of mesoderm spreading in Drosophila embryos: Activation of MAPK

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).

Nuclear localization of the ERK MAP kinase mediated by Drosophila alphaPS2betaPS integrin and importin-7

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants (see Myospheroid), either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7-deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein (James, 2007).

The integrin cell surface receptors regulate numerous cellular processes, including growth, differentiation, apoptosis and migration. Integrins are heterodimers made up of α and β subunits, each with short cytoplasmic tails and large extracellular domains. Integrins function as adhesion molecules and frequently form a physical connection between the extracellular matrix (ECM) and the actin cytoskeleton (James, 2007).

In addition to their function in cell adhesion, integrins are critical to many of the signaling pathways of cells. Of particular relevance to these studies, numerous examples have been documented in which integrins regulate the activity of mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK), or in turn are regulated by these enzymes. Integrins may directly mediate ERK activation, or in other cases, they may function to modulate the activities of growth factor receptors on ERK signaling (James, 2007).

ERK-induced gene expression requires the transport of ERK into the nucleus. In the absence of stimulation, ERK is maintained in the cytoplasm through an interaction with its upstream activator mitogen-activated protein kinase kinase (MEK). MEK phosphorylates both tyrosine and threonine residues in the activation loop of ERK. After phosphorylation by MEK, diphospho-ERK (dpERK) probably dimerizes and enters the nucleus via an active transport mechanism. The subcellular localization of dpERK after activation offers an additional level of regulation of ERK signaling (James, 2007 and references therein).

In general, cells in suspension respond weakly to growth factor stimulation compared with cells adhering to the ECM, and regulation of ERK nuclear import is one potential step where integrin and receptor tyrosine kinase (RTK) signals may be integrated. For example, after activation by MEK in NIH 3T3 cells maintained in suspension the majority of ERK remains in the cytoplasm, and ERK activation of the transcription factor Elk-1 is reduced compared with adherent cells. A β4 integrin signaling domain has been shown to affect the nuclear translocation of MAP kinases and NF-kappaB although the large cytoplasmic domain of β4 is not homologous to that of other integrin β subunits. The nuclear localization of other transcriptional regulators also has been shown to be altered by integrin function in mammalian cells, including the c-Abl tyrosine kinase in mouse fibroblasts and the transcriptional coactivator JAB1 in a variety of cell types. Additional connections between integrins and nuclear import are suggested by studies on proteins that are typically considered to be downstream of integrins. For example, integrin-linked kinase (ILK) has been shown to regulate the nuclear import of a c-Jun coactivator protein (James, 2007).

A potential link between integrins and nuclear import has been further suggested by studies of wing development in Drosophila. In Drosophila, integrins are required to maintain the attachment of the dorsal and ventral wing epithelia during adult morphogenesis, and this process depends on the differential expression αPS1 and αPS2 integrin subunits on the dorsal and ventral cells, respectively. Loss of integrin function leads to wing blisters, where the two surfaces separate after eclosion of the adult from the pupal case. Surprisingly, wing blisters can also occur when an α subunit is inappropriately expressed on the wrong side of the wing, and experiments with various mutants have demonstrated that this is a gain-of-function phenotype. That is, the activity of an integrin in the wrong place during a specific morphogenetic event causes a subsequent loss of epithelial attachment. A genetic screen for dominant suppressors of this gain-of-function wing blister phenotype (Baker, 2002) identified null mutations in a gene named moleskin (msk) (James, 2007).

The moleskin gene encodes Drosophila Importin-7 (DIM-7), which is a close homologue of vertebrate Importin-7, also known as Ran Binding Protein-7 (RanBP-7). Importin-7 is a member of the importin β superfamily of nuclear importers, which can bind directly to the nuclear pore complex. Vertebrate Importin-7 has been shown to mediate nuclear import of ribosomal proteins, histone H1, the HIV-1 reverse transcription complex and the glucocorticoid receptor. In Drosophila, DIM-7 is tyrosine phosphorylated in response to growth factor stimulation of RTKs, and it physically binds Drosophila ERK. Additionally, DIM-7 binds the tyrosine phosphatase Corkscrew (CSW), the Drosophila homologue of SHP-2. Corkscrew is generally required for ERK signaling via RTKs, and in vertebrate cells SHP-2 has been associated with integrin signaling and regulation of integrin activity, although the molecular bases of these interactions remain unclear (James, 2007).

Until recently, it has not been clear how dpERK gains entry to the nucleus after activation. In addition to regulated nuclear import, the cellular localization of phosphorylated ERK dimers can be influenced by release from cytoplasmic anchors and regulated nuclear retention or export, and in at least one case it has been suggested that ERK2 may not require any additional import proteins. Genetic experiments with Drosophila embryos demonstrate that DIM-7 is largely responsible for the nuclear import of activated ERK in this system. The suppression of integrin-related phenotypes in fly wings by moleskin mutations led to an examination a potential connection between integrins and the regulation of ERK import in a Drosophila cell culture system, and the results suggest that DIM-7 may represent a novel nexus of integrin and RTK signaling (James, 2007).

This study shows that a vertebrate homologue of DIM-7 can rescue the ERK localization phenotype of DIM-7 dsRNA treated cells. Thus, ERK nuclear translocation is a property of members of the Importin-7 family of proteins generally. This function cannot necessarily be extended to other MAP kinases; for example, no change is seen in nuclear localization of the p38 MAP kinase in S2 cells grown in DIM-7 dsRNA, although c-Jun NH2-terminal kinase transport does seem to involve DIM-7 (James, 2007).

The ability of growth factors to activate ERK signaling is often linked to integrins; however, specific integrin functions typically have not been examined. The reduced levels of nuclear dpERK in cells expressing βPS-G1 (which contains a frameshift mutation in the cytoplasmic domain that eliminates the two NPXY motifs that are critical for interaction with a number of cytoplasmic proteins, including talin) or βPS-G4 (which has a mutation in the second serine of the MIDAS domain (DXSXS), which would be expected to inhibit extracellular ligand binding) show that ERK signaling seen in Drosophila S2 cells is dependent on functional integrins and that it is not due solely to changes in cell adhesion or shape. Specifically, both the extracellular and cytoplasmic integrin domains must be able to interact properly with ECM or intracellular components for the integrins to support high levels of nuclear dpERK (James, 2007).

A simple model in which soluble dpERK (activated by integrins or growth factors) finds DIM-7 ('activated' by integrins) for import cannot explain all of the data. The experiments that examine total ERK distribution in the integrin mutants suggest that most of the activated ERK cannot enter the nucleus even after translocation into the DIM-7-rich perinuclear region. One would expect that the importins here are actively working with various other cargos and that a significant fraction of the DIM-7 is generally capable of import. This result suggests a model in which ERK phosphorylation must be spatially coupled to DIM-7 activation to make a complex that can be imported efficiently (see Model for the role of integrins in ERK nuclear import in S2 cells). Interestingly, a specific membrane localization of DIM-7 has been suggested as a regulatory mechanism in developing Drosophila eyes (Vrailas, 2006); however, in this case the targeting to apical epithelia has been seen as an inhibitory mechanism (James, 2007).

moleskin (DIM-7) function is required for normal cell proliferation in animals, where patches of mutant cells disappear in developing epithelia. Examples in which a 50% reduction in DIM-7 function has produced phenotypes in developing flies have involved circumstances in which a signaling pathway has been stimulated to high levels by gene overexpression. In the current study DIM-7 levels are reduced significantly, but they are not eliminated, and the cells show no obvious phenotype during normal growth. However, clear effects are seen after acute stimulation of the ERK pathway. The integrin-mediated activation of DIM-7 may be especially important as a regulatory component in such cases of acute, high-level signaling (James, 2007).

Mouse embryo fibroblasts expressing a β1 mutant similar to βPS-G1 transiently display elevated phospho-ERK after stimulation with growth factor, but subsequently the same groups reported that ERK does not necessarily enter the nucleus after plating on fibronectin. Interestingly, the import defect seen in the integrin mutants can be rescued by adding constitutively active Rac. Although multiple pathways may couple integrins to ERK activation and transport in different cell types, Importin-7 family members are likely to be a common feature of dpERK nuclear translocation, and it will be interesting to see whether various pathways leading from integrins to import converge at this protein (James, 2007).

Perhaps most intriguing with respect to the current studies is the work on a natural human β1 variant. β1C is an alternatively spliced form that, like βPS-G1, replaces the cytoplasmic NPXY motifs with other sequence. Cells that express β1C show reduced proliferation and reduced activation of the Ras-ERK pathway, relative to cells expressing the more common β1A. β1A-containing integrins seem to form a complex that includes insulin-like growth factor-I receptor and the insulin receptor substrate-1 (IRS-1), and the addition of insulin leads to cell proliferation and inhibition of adhesion to laminin. In contrast, β1C expression leads to decreased proliferation and increased adhesion, and these effects seem to be mediated by a complex that includes Gab1 and SHP-2, but not IRS-1. There is no β1C variant naturally in Drosophila, but the βPS-G1 mutant does show some dominant-negative effects in flies, and the work reported in this study suggests that this might result at least in part from disruptive effects on intracellular signaling (James, 2007 and references therein).

Significant amounts of cortical DIM-7 are found where integrins are located at the spreading edges of cells. Consistent with a role of integrins in peripheral DIM-7 localization, cells that are protease treated, heat shocked to induce integrin expression, and spread in serum-free media (where spreading is integrin independent), DIM-7 is not found at the periphery in fully spread cells at early times, but it appears when integrin expression is detected after a few hours. One striking feature of the peripheral DIM-7 is that it does not colocalize with integrins in more organized cell-substratum adhesion sites. Thus, peripheral integrin-DIM-7 associations seem to depend on the functional state of the integrins (James, 2007).

Vertebrate SHP-2 is necessary in many contexts for growth factor activation of ERK, and SHP-2 has also been shown to be involved in integrin-dependent signaling. However, the data from different cell types fail to paint a simple, cohesive picture of SHP-2 molecular function, especially with respect to signaling downstream of integrins. Previous biochemical data indicate that DIM-7 binds Drosophila Corkscrew (SHP-2) (Lorenzen, 2001). Corkscrew is generally an essential component in signaling via receptor tyrosine kinases; however, Corkscrew lacking tyrosine phosphatase activity can rescue some phenotypes when reintroduced into corkscrew mutants, suggesting that in some contexts Corkscrew functions primarily as a scaffolding protein. The role of Corkscrew in DIM-7 activation may be largely as a scaffold, because DIM-7 disappears from the periphery when Corkscrew is depleted. This does not seem to be an indirect result of a defect in DIM-7 activation, because cortical DIM-7 remains in other situations that affect its ability to import dpERK, for example, in both of the integrin mutants tested (James, 2007 and references therein).

Interestingly, the screen that identified moleskin (DIM-7) as a suppressor of Blistermaker assayed only ~40% of the Drosophila genome (the third chromosome). Further elucidation of the molecular mechanisms underlying the DIM-7/integrin connection is likely to be facilitated by the identification of additional Blistermaker suppressors on other chromosomes. Screens for such loci are in progress (James, 2007).

Unc-51 controls active zone density and protein composition by downregulating ERK signaling

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. This study used a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 (see Atg1) acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability (Wairkar, 2009).

A large-scale anatomical screen was performed to identify mutants where not every glutamate receptor cluster is apposed to Bruchpilot. Mutants with a global decrease in Brp or DGluRIII across the NMJ were put aside, and instead focus was placed on mutants in which Brp was absent from a subset of synapses. Such mutants were identified by the presence of glutamate receptor clusters unapposed to Bruchpilot puncta. In this screen, mutants were identified in unc-51 (Wairkar, 2009).

In the unc-51 mutant many DGluRIII clusters are unappposed to Brp. Such misapposition could reflect either DGluRIII clusters unapposed to active zones, or receptor clusters apposed to abnormal active zones that do not contain Brp. The ideal experiment to distinguish between these possibilities would be to stain for other presynaptic active zone proteins. Unfortunately the only other such protein that can be visualized in Drosophila is the calcium channel Cacophony, and since its localization depends on Brp this experiment is not be informative. Nonetheless, two results strongly suggest that a subset of glutamate receptors is apposed to abnormal active zones. First, the decreased density of DGluRIII clusters observed via confocal microscopy approximates the decrease in active zone density observed via electron microscopy. If many DGluRIII clusters were unapposed to active zones, then a more dramatic decrease in active zone density would be expected. Second, ultrastructural analysis demonstrates a decrease in the proportion of active zones containing T-bars. Brp is not necessary for the formation of active zones, but is required for the localization of T-bars to active zones. If the absence of Brp were due to the absence of the entire active zone, then each active zone would contain Brp and a normal ratio of T-bars/active zones would be predicted. Instead, the decrease in T-bars/active zone is consistent with the presence of active zones missing Brp and, hence, lacking T-bars. Therefore, it is concluded that Unc-51 is required for the high fidelity of active zone assembly, ensuring that Brp is present at every active zone (Wairkar, 2009).

In addition to the presence of abnormal synapses in the unc-51 mutant, there is also a decrease in the number and density of synapses. It is speculated that the decrease in synaptic density and the presence of abnormal synapses may be related phenotypes that differ in severity. In this view, Unc-51 promotes synapse formation. In its absence, active zone assembly would be less efficient, resulting in either the formation of abnormal active zones missing crucial proteins such as Brp, or in more severe cases leading to complete failure of active zone assembly and, hence, the absence of a synapse. The complete suppression of both the synaptic density and apposition phenotypes by mutation of the downstream target ERK is consistent with these phenotypes sharing an underlying mechanism. As expected, this defect in the number and proper assembly of synapses leads to a dramatic decrease in synaptic efficacy (Wairkar, 2009).

In addition to these synaptic defects, the unc-51 mutant also has a smaller NMJ and accumulations of synaptic material in the axons, suggesting defects in axonal transport. One mechanism that could link a small NMJ with defective transport is synaptic retraction, in which entire presynaptic boutons or branches retract leaving a footprint of postsynaptic proteins. However, no such footprints were observed in the unc-51 mutant, so this is not the cause of the small NMJ. Synaptic growth requires the retrograde transport of a BMP signal to the nucleus, however this study no change in the levels of phosphorylated MAD in motoneuron nuclei, suggesting that this is not a likely cause of the growth defect. Finally, in worms and mice Unc-51 is required for axon outgrowth, which may be somewhat analogous to defects in NMJ growth in Drosophila. However, to form an NMJ the axon must navigate out of the ventral nerve cord and cross a wide expanse of muscle before reaching its target and forming a junction. Since no defects were observed in the pattern of neuromuscular innervation, it is unlikely that a generic defect in axon outgrowth is responsible for the small NMJs. The apparent axonal transport defect is consistent with findings from mammals suggesting a function for Unc-51 in regulating axon transport. The role of Unc-51 for transport was not investigated, but note that it was possible to genetically separate the axonal transport and synapse development phenotypes, so the transport phenotypes may not be primary cause of the synaptic defects (Wairkar, 2009).

These data support the model that Unc-51 inhibits ERK activation to promote proper active zone development. In the unc-51 mutant a modest increase was observed in the levels of activated ERK, demonstrating that Unc-51 is a negative regulator of ERK activation in vivo. This increased ERK activity is responsible for the defects in active zone formation. Double mutants between unc-51 and the ERK hypomorph rl1 completely suppress the synapse density and apposition phenotypes of the unc-51 mutant, and restore synaptic strength to wild type levels. Hence, ERK is required for the synaptic phenotypes observed in the unc-51 mutant. The axonal transport defects were not suppressed in the double mutant, so Unc-51 must act through other pathways as well. In mammalian cells Unc-51 can downregulate ERK by inhibiting the binding of a scaffolding protein to the FGF receptor. To date, no receptor tyrosine kinase has been identified that regulates active zone formation in Drosophila. Future studies to characterize the mechanism by which Unc-51 inhibits ERK in Drosophila motoneurons may provide clues towards identification of such a pathway. In addition, it is unclear how ERK regulates active zone formation. A previous study demonstrated that phospho-ERK localizes to the active zone, which would suggest a direct mechanism. Unfortunately, these localization findings could not be replicated. The same study demonstrated that the transgenic expression of a constitutively active ras or a gain-of-function ERK allele both lead to an increase in the number of synaptic boutons, which is not consistent with the current finding of a smaller NMJ. Active zone structure and number were not assessed. It is speculated that the global activation of ERK may result in different phenotypes than relief of Unc-51 inhibition of ERK, which could show temporal and spatial specificity (Wairkar, 2009).

In mammalian and Drosophila neurons, release probability varies across release sites formed by a single neuron. One potential mechanism would be the differential localization or activity of core active zone proteins. In Drosophila, Bruchpilot is an excellent candidate for such a protein. It is required for the localization of calcium channels to the active zone, so changes in its localization or function would impact calcium influx and, hence, release probability at an active zone. The unc-51 mutant demonstrates that signaling pathways can differentially regulate the localization of Brp to individual release sites within a single neuron. As such, the Unc-51/Erk signaling pathway is a candidate mechanism to regulate active zone protein composition and release probability in a synapse-specific manner (Wairkar, 2009).

beta-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. This study reports a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. These findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz (Tipping, 2010).

This study demonstrate that the Krz protein is necessary for setting a precise level of activation of two maternal signalling pathways, Torso and Toll. This activity of Krz helps to establish the correct domains of expression of developmental patterning regulators that are under the control of these pathways (Tipping, 2010).

Genetic and protein interaction data suggest a new mechanism by which Krz may limit the activity of Torso. It was observed that Krz preferentially binds and sequesters an inactive form of ERK, thereby making it unavailable for activation by the upstream kinases such as MEK. Such a mechanism of direct inhibition of ERK activation by β-arrestin binding has not been previously reported. This mechanism is consistent with the observed in vivo effects of loss of krz on ERK activity. In krz maternal mutant embryos, ERK is not sequestered and therefore more ERK is available to transduce Torso signals, resulting in hyperactivation of Torso target genes, tll and hkb. Furthermore, consistent with this model is the observation that Krz and MEK apparently compete for ERK when all three proteins are co-expressed in S2 cells (Tipping, 2010).

Interaction assays using mutated forms of Krz and ERK indicate that the conformations of both proteins have an effect on their binding affinity. On binding to an activated GPCR, the arrestin molecule undergoes a dramatic conformational change that can be mimicked by specific mutations (Gurevich, 2004). In immunoprecipitation experiments it was observed that such 'pre-activated' form of Krz (R209E) has a much greater affinity for ERK, compared with the wild-type Krz protein, and that this higher affinity is also observed for the equivalent mutant of human β-arrestin2. This suggests that the ERK-binding ability of β-arrestin may be affected by its conformation, but it is unknown at present whether any upstream signals convert Krz into an activated form in the embryo. Overexpression of Krz-R209E using the da-GAL4 driver did not result in any observable phenotype and could rescue zygotic loss of krz, suggesting that it retains most of the functions of wild-type Krz (data not shown) (Tipping, 2010).

It was observed that the conformation of ERK itself has a large effect on its interactions with Krz. In the binding experiments, activated forms of ERK bind Krz (and human β-arrestin2) with lower affinity, compared with wild-type inactive ERK. Moreover, mutations in the TEY motif, which render ERK constitutively inactive, also lower its affinity for Krz, which is at a first glance a surprising result. However, previous studies have shown that both types of mutations in the TEY motif, which is a part of the activation loop, increase disorder in the lip region and cause a conformational change in the ERK molecule that makes it different from the basal state. It is therefore speculated that the activation loop may be involved in mediating an interaction of ERK with β-arrestin. Consistent with the current results, deviation of ERK structure from the basal state would decrease its association with β-arrestin (Tipping, 2010).

Other studies have reported formation of protein complexes containing β-arrestins and an activated form of ERK. It is possible that in those experimental conditions other binding partners, such as Raf or the activated receptor, assist in stabilizing the complex of MAP kinases with β-arrestin. This study has shown that although Krz can bind to the Drosophila homologues of both MEK and Raf, overexpression of Krz does not increase production of dpERK by the MAPK cascade downstream of activated RTKs, but instead appreciably inhibits it in the absence of overexpressed Raf. The data do not rule out a possibility that Krz may still promote ERK activation in other biological contexts, particularly downstream of activated GPCRs, but this question awaits further investigation (Tipping, 2010).

Interestingly, the sequestration mechanism of ERK inhibition described in this study is different from the effects of Krz on Notch. Previous studies have shown that Krz inhibits Notch activity by forming a ternary complex with Deltex and the Notch receptor. Formation of this complex increases Notch turnover and thereby downregulates Notch signalling (Mukherjee, 2005). No change was observed in ERK turnover in the presence of wild-type overexpressed Krz, suggesting that Krz is unlikely to be involved in the regulation of ERK stability. However, given the versatility of molecular functions displayed by β-arrestins, it is possible that there are other, as yet uncharacterized mechanisms by which Krz controls signalling downstream of RTKs (Tipping, 2010).

The inhibitory effects of Krz on ERK activity are not limited to the Torso pathway and early embryogenesis, but are also observed in other tissues and at later developmental stages. Thus, broadening of the dpERK patterns activated by EGFR and Btl was observed in krz maternal mutant embryos. An increase in the overall levels of dpERK during mid-to-late embryogenesis was also detected on western blots. Later in development, ERK is activated by EGFR in the wing and both EGFR and Sevenless in the eye. Genetic data suggest that Krz also inhibits ERK activity in these tissues during larval development. A broad involvement of Krz in inhibiting ERK activity suggests that Krz has a general inhibitory role to limit the activity of different RTKs in Drosophila development (Tipping, 2010).

In addition to its effects on RTK signalling, it was observed that Krz has an important role in limiting the activity of the Toll receptor, which specifies the development of the ventral structures. Other studies have reported that mammalian β-arrestins can downregulate NF-κB signalling by binding and stabilizing the NF-κB inhibitor IκBα. The inhibitory effects of Krz on Dorsal may involve a similar mechanism. It was observed that Krz can directly bind to the Drosophila orthologue of IκBα, Cactus, suggesting that the mechanism of NF-κB inhibition by β-arrestins at the level of IκBα may be conserved. Consistent with this finding, a decrease was detected in the level of the Cactus protein in krz maternal mutants at 0-4 h of development, which may explain the observed expansion of the nuclear gradient of Dorsal in these mutants. It is still unclear why expansion of Dorsal nuclear localization is more pronounced in the posterior half of the embryo (Tipping, 2010).

In the developing embryo, the Torso and Toll pathways do not work in isolation, but are involved in cross-regulatory interactions on certain common targets, such as zen. zen is repressed by nuclear Dorsal in the ventral part of the embryo, and relieved of this repression (de-repressed) by the signalling activity of Torso emanating from the embryo poles. The molecular mechanism of this de-repression is still unknown. It was observed that loss of krz shifts the balance of the effects of Torso on Toll, which results in an inappropriate expansion of zen expression at the embryo poles. It is speculated that Krz helps Torso to achieve a precise level of de-repression of zen by limiting the activity of ERK. Krz is thus able to control the separate activities of the Torso and Toll pathways (reflected in its effects on tll, hkb, twi, and rho), as well as regulate common Torso and Toll targets such as zen. For such pathways that are engaged in cross-regulatory interactions, Krz ensures that a proper level of signalling activity from one pathway reaches the other. This function adds an important new mechanism to understanding of the ways in which signalling pathways are coordinately regulated during development (Tipping, 2010).

A ubiquitous distribution of Krz in the embryo agrees with the dysregulation of multiple pathways observed in krz mutant animals. As overexpression of Krz does not cause any obvious defects, the level of Krz itself is not limiting for the regulation of signalling. Instead, Krz apparently makes other signalling co-factors limiting for their respective pathways, essentially working as a molecular 'sponge' to prevent pathway hyperactivity. Specificity of Krz function is likely to be determined by its selective interactions with specific pathway co-factors. Maternal loss of krz function thus affects multiple developmental signalling pathways, resulting in an accumulation of defects that ultimately lead to severe morphological abnormalities such as a disruption of gastrulation movements. By analysing the effects of loss of krz on individual pathways in vivo, this study has been able to show its role in the regulation of RTK and Toll signalling. Future studies will likely reveal other pathways and levels of regulation that are under the control of the Drosophila β-arrestin Krz (Tipping, 2010).

MAPK-dependent phosphorylation modulates the activity of Suppressor of Hairless in Drosophila

Cell differentiation strictly depends on the epidermal growth factor receptor (EGFR)- and Notch-signalling pathways, which are closely intertwined. This study addresses the molecular cross talk at the level of Suppressor of Hairless [Su(H)]. The Drosophila transcription factor Su(H) mediates Notch signalling at the DNA level: in the presence of signalling input Su(H) assembles an activator complex on Notch target genes and a repressor complex in its absence. Su(H) contains a highly conserved mitogen activated protein kinase (MAPK) target sequence. Evidence is provided that Su(H) is phosphorylated in response to MAPK activity. Mutation of the Su(H) MAPK-site modulated the Notch signalling output: whereas a phospho-deficient Su(H)MAPK-ko isoform provoked a stronger Notch signalling activity, a phospho-mimetic Su(H)MAPK-ac mutant resulted in its attenuation. In vivo assays in Drosophila cell culture as well as in flies support the idea that Su(H) phosphorylation affects the dynamics of repressor or activator complex formation or the transition from the one into the other complex. In summary, the phosphorylation of Su(H) attenuates Notch signalling in vivo in several developmental settings. Consequently, a decrease of EGFR signal causes an increase of Notch signalling intensity. Hence, the antagonistic relationship between EGFR- and Notch-signalling pathways may involve a direct modification of Su(H) by MAPK in several developmental contexts of fly development. The high sequence conservation of the MAPK target site in the mammalian Su(H) homologues supports the idea that EGFR signalling impacts on Notch activity in a similar way in humans as well (Auer, 2014).

The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development

Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined in this study. Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. These data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling (Mouchel-Vielh, 2011).

The ectopic vein phenotype of corto mutants was investigated using three different recessive lethal alleles: corto420, corto07128b and cortoL1. corto420 is a deletion of the corto locus, corto07128b a P-element insertion located 0.5 kb upstream of corto 5'-UTR, and cortoL1 an EMS-induced mutation. Heteroallelic combinations using corto07128b, cortoL1 and a deficiency encompassing corto [Df(3R)6-7] are poorly viable, since 0% to 10% escapers were observed depending on combinations. Therefore, these three alleles are true loss-of-function alleles. This was confirmed by quantitative RT-PCR analysis on wing discs from third instar larvae, that showed absence of corto transcripts in corto420/Df(3R)6-7 and corto07128b/Df(3R)6-7 larvae. In contrast, cortoL1/Df(3R)6-7 larvae exhibited the same level of corto transcripts as wild-type flies, which suggests that the mutation in cortoL1 rather affects the level or activity of Corto protein (Mouchel-Vielh, 2011).

corto420/+ heterozygous flies exhibited very few ectopic veins (2.2% to 8.6%). This phenotype was more penetrant in cortoL1/+ (49% to 52.2%) and corto07128b/+ (97.4% to 97.8%) heterozygous flies. Since both corto420 and corto07128b are devoid of corto transcripts, the discrepancy between these alleles may be a consequence of an interaction with the genetic background. For all combinations, the few heteroallelic corto escapers displayed a stronger ectopic vein phenotype than corto heterozygous flies. Ectopic veins mainly arose close to longitudinal veins 2, 3, 5 and to the posterior cross-vein, which seems to be the case for most mutations that induce ectopic vein phenotypes. Interestingly, over-expressing corto using a UAS::corto construct and the wing specific Beadex::Gal4 (Bx::Gal4) or scalloped::Gal4 (sd::Gal4) driver also induced extra pieces of vein tissue in all flies. Since both corto over-expression and loss-of-function induced the same phenotype, one possibility is that Corto may be required in stoechiometric amount to allow correct wing tissue differentiation. This feature characterizes proteins that act through formation of complexes. Indeed, complexes are very sensitive to the relative amounts of their components, and can be disrupted either by an excess or a shortage of one of these (Mouchel-Vielh, 2011).

In order to assess the temporal requirement for corto function in wing tissue differentiation, the UAS::corto line was crossed with the hs::Gal4 driver strain allowing staged Gal4 expression. The highest percentage of ectopic vein phenotype was obtained when heat-shock was applied between 96 to 120 hours after egg laying, which corresponds to the mid to late third instar larval stage. Interestingly, it has been shown that, from late third instar larval stage to pupal stage, down-regulation of ERK signaling is crucial for wing tissue formation: indeed, expression of a constitutively active form of the MAPKK Raf at the third instar larval stage induces vein loss, whereas expression of a dominant negative form of the receptor DER at pupal stage leads to formation of ectopic veins (Mouchel-Vielh, 2011).

In conclusion, corto misregulation (either loss-of-function or over-expression) induced ectopic veins that formed within intervein tissue and never truncated veins. This observation suggested that Corto contributes to intervein tissue differentiation, whereas it does not seem to be involved in vein formation. It was previously shown that corto interacts with some TrxG genes during wing tissue formation. Indeed, moira, kismet and ash1 mutants enhance the ectopic vein phenotype of corto420. Furthermore, several corto alleles enhance the ectopic vein phenotype of mutations in snr1 that encodes a component of the SWI/SNF complex, a chromatin-remodeling complex also involved in wing tissue differentiation. One hypothesis is that Corto, as an ETP, could participate in the recruitment of TrxG complexes to regulate expression of genes involved in wing tissue differentiation (Mouchel-Vielh, 2011).

To clarify the role of corto in the formation of intervein tissue, genetic interaction assays were performed between corto and the intervein-promoting gene blistered (bs), or the vein-promoting gene rhomboid (rho). As expected for a bs loss-of-function allele, wings of flies heterozygous for bsEY23316 exhibited a moderate ectopic vein phenotype, but none showed blisters in the wings. corto07128b enhanced the ectopic vein phenotype induced by bsEY23316. In addition, 32.5% of these trans-heterozygous flies had blisters in the wings. These blisters, which result from impaired adhesion between the ventral and dorsal wing surfaces, could be caused by formation of many vein cells within intervein tissue. They are frequently observed in bs mutants or when rho is over-expressed. This result therefore showed that bs and corto act synergistically to promote intervein cell fate. Ectopic over-expression of rho using the rhoEP3704 allele and the sd::Gal4 driver induced ectopic veins for most of the flies and in a few cases (9.5%) formation of blisters. This phenotype was similar to that induced by over-expressing rho under control of a heat-inducible promoter. Both corto420 and corto07128b alleles enhanced this phenotype since the number of flies with blisters in the wings significantly increased. This observation showed that corto antagonizes rho in vein formation (Mouchel-Vielh, 2011).

Taken together, these results suggest that corto might antagonize rl vein-promoting function in future intervein cells. corto misregulation could therefore lead to deregulation of certain vein and intervein-promoting genes. Indeed, deregulation of bs and rho was observed in some intervein cells of pupal wings from cortoL1/Df(3R)6-7 escapers: in these cells, bs is down-regulated whereas rho is ectopically expressed. These cells could thus acquire a vein fate (Mouchel-Vielh, 2011).

Since the wing phenotype of corto mutants resembles the one induced by hyperactivation of ERK signaling pathway, it was asked whether corto was involved in the regulation of this pathway during wing development. Genetic interactions between corto and rolled were tested using the UAS::rolled strain which allows targeted ERK over-expression when crossed with a Gal4 driver. All flies over-expressing rolled with the sd::Gal4 driver at 25°C exhibited a mild ectopic vein phenotype. Expressivity of this phenotype was enhanced by the corto07128b allele. This result suggests that the roles of corto and rolled in vein-promoting function are antagonistic (Mouchel-Vielh, 2011).

The UAS::rolledSem (rlSem) transgene that encodes a hyper-active form of ERK was used. At 18°C, flies that over-expressed the UAS::rlSem transgene under control of the sd::Gal4 driver exhibited ectopic veins. This phenotype was much stronger than the one induced by rolled over-expression. For 88% of these flies, this phenotype was very strong since one or the two wings showed blisters. Surprisingly, penetrance and expressivity of the rlSem over-expression phenotype were lowered by corto420 and corto07128b alleles, as only 49.4% of corto420 flies and 55.1% of corto07128b ones exhibited blisters in one or both wings and blisters were smaller. This result confirmed that corto and rl interact during wing tissue formation. However, the observation that corto mutation enhanced a mild-activation of ERK pathway (as induced by UAS::rl) whereas slowing-down a hyper-activation (as induced by UAS::rlSem) is paradoxical and requires further experiments to be fully understood (Mouchel-Vielh, 2011).

It has recently been shown that the Drosophila ortholog of MP1, dMP1, antagonizes rl vein-promoting function in the future intervein cells of the wing (Mouchel-Vielh, 2008). Furthermore, dMP1 was isolated in a two-hybrid screen using Corto as bait. Thus the genetic interactions between corto and dMP1 was tested. Down-regulation of dMP1 by RNA interference using the sd::Gal4 driver induced ectopic veins in 78.5% of flies. This percentage increased to 92.2% and 100% in combination with corto420 or corto07128b, respectively. With corto07128b, the expressivity of the ectopic vein phenotype was also enhanced. Therefore, these results showed that corto and dMP1 act synergistically and participate in intervein tissue differentiation in response to ERK signaling (Mouchel-Vielh, 2011).

dMP1 forms a complex with ERK, which is required for the proper development of intervein cells. To understand the molecular bases of the relationship between Corto, dMP1 and ERK, the physical interaction between Corto and ERK was examined. GST pull-down assays were performed using in vitro translated ERK and GST-Corto fusion proteins. Structural analysis of Corto has shown that this 550 amino-acid protein contains three globular domains that might correspond to functional domains. The first one is located at position 127-203 and exhibits strong structural similarities with chromodomains, that are chromatin targeting modules found in some regulators of chromatin structure. The two others, located at positions 418-455 and 480-550, present no obvious similarities with known protein domains. In vitro translated ERK protein was retained on GST-C1/324 and GST-C325/550 beads containing the NH2-terminal half and the COOH-terminal half of Corto, respectively. In contrast, ERK was not retained on GST-C127/207 beads containing the Corto chromodomain, or on GST-C418/503 beads containing part of the two COOH-terminal globular domains. The lack of interaction with GST-C127/207 and GST-C418/503 suggested that none of these domains was sufficient to mediate Corto-ERK interaction, either because of inappropriate folding of these short domains in the GST fusion proteins, or because none of these two fragments contains the sequences that mediate ERK binding. Taken together, these results showed that Corto interacts directly with ERK in vitro. Further experiments are needed to determine the precise domains or residues that mediate the interaction between Corto and ERK (Mouchel-Vielh, 2011).

Since dMP1 was isolated in a two-hybrid screen using the NH2-terminal part of Corto as a bait, the physical interaction between Corto and dMP1 was examined. GST pull-down assays were performed using GST-dMP1 fusion protein and in vitro translated Corto to see whether their interaction was direct or indirect. Indeed, indirect interactions via yeast proteins have already been observed in two-hybrid experiments. The same result was obtained using GST or GST-dMP1 beads indicating that there was no specific direct interaction between Corto and dMP1. However, by incubating GST-dMP1 beads with total embryonic protein extract, Corto was specifically retained on GST-dMP1 beads. Therefore, it is concluded that Corto and dMP1 interact via additional factors. One potential candidate could be ERK, since it directly interacts with Corto and with dMP1 (Mouchel-Vielh, 2011).

This study has showm that the ETP corto, rl and dMP1 interact during wing tissue differentiation in Drosophila. Corto, ERK and dMP1 form a complex exclusively in the nucleus. In addition, these proteins bind polytene chromosomes where they partially co-localize, suggesting that the Corto-ERK-dMP1 complex might regulate vein and/or intervein gene expression directly on chromatin. Future experiments will be needed to test whether this complex, via the ETP Corto, participates in the recruitment of TrxG complexes on target genes in response to ERK signaling (Mouchel-Vielh, 2011).

Neuronal Cbl controls biosynthesis of insulin-like peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. This study shows that in Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. dCbl is highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increases sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl results in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulates the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolishes the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increases insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells (Yu, 2012).

Tay bridge is a negative regulator of EGFR signalling and interacts with Erk and Mkp3 in the Drosophila melanogaster wing

The regulation of Extracellular regulated kinase (Erk) activity is a key aspect of signalling by pathways activated by extracellular ligands acting through tyrosine kinase transmembrane receptors. Proteins with kinase activity that phosphorylate and activate Erk, as well as different phosphatases that inactivate Erk by de-phosphorylation, participate in this process. The state of Erk phosphorylation affects not only its activity, but also its subcellular localization, defining the repertoire of Erk target proteins, and consequently, the cellular response to Erk. This work characterises Tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge is a large nuclear protein with a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. This study shows that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mitogen-activated protein kinase phosphatase 3 (Mkp3). It is suggested that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus (Molnar, 2013).

Signalling by Erk in response to growth factors regulates growth, differentiation and survival of cells in a variety of developmental contexts. The extent and level of Erk activation relies on its phosphorylation state, which in turns regulates Erk subcellular localization and interactions with downstream effectors and other proteins. Erk activation is transient, and failures in the mechanisms responsible for its inactivation can drive developmental defects and oncogenic transformations. This work identified Tay as a novel nuclear component that interacts with Erk and is involved in the maintenance of appropriate levels of Erk activity (Molnar, 2013).

This study has addressed the requirements and function of tay mostly in the wing disc, a convenient developmental system to analyse the contribution of signalling pathways to the regulation of organ size and pattern formation. Tay was previously described as a protein that regulates locomotion and other neural aspects (Poeck, 2008). This study has seen that changes in the level of EGFR signalling in the nervous system also cause locomotion defects, which is indicative of a role of Tay in the regulation of EGFR signalling also in the nervous system. In the context of wing development and vein differentiation, the loss of tay results in the differentiation of extra veins in inter-vein territories. This phenotype is very similar to those obtained in conditions of excess of EGFR signalling, suggesting that Tay negatively regulates the activity or the response to this pathway. In addition, loss of tay also causes a reduction in the size of the wing blade, a phenotype that is not expected in a situation of excess of EGFR/ERK activity. This last result suggests that Tay might also have functions independent of its role in the regulation of EGFR signalling. The consequences of gain of Tay expression mostly indicate that the role of Tay is related to the modulation of EGFR signalling. Thus, excess of Tay expression in different imaginal discs results in phenotypes that can be attributed to loss of EGFR signalling, such as loss of veins and bristles, wing size reduction and failures in tarsal joint formation and ommatidial differentiation (Molnar, 2013).

This study further explore the relationships between Tay and EGFR signalling in genetic combinations in which the activity of the pathway is altered in backgrounds with modified levels of Tay expression. In all cases, synergistic interactions were observed between loss of tay and excess of EGFR, and between excess of tay and loss of EGFR activity. Furthermore, it was noticed that the extra veins differentiating in tay mutants require EGFR function, suggesting that Tay modulates EGFR signalling during vein formation. All together, the results of genetic combinations indicate that cells with lower levels of Tay become more sensitive to an increase in EGFR signalling, and that Tay over-expression prevents cells to acquire the level of EGFR signalling required for vein formation (Molnar, 2013).

The negative effect of Tay on EGFR signalling is more directly visualised by considering the effects of Tay in Erk phosphorylation and in the expression of the EGFR/Erk targets genes Dl and argos. Thus, Tay over-expression strongly suppresses Erk phosphorylation and prevents the expression of Dl and argos in the developing veins. Conversely, in loss of tay conditions an increase was detected in the levels of phosphorylated Erk, which is accompanied by a moderate ectopic expression of argos. The extra-vein phenotype of loss of tay is not as extreme as the massive vein differentiation that occurs upon strong and constitutive activation of the EGFR pathway. In fact, tay mutant wings differentiate a similar pattern of extra veins as moderate increases in EGFR signalling caused by, for example, mutations in the Mkp3 gene (Gomez, 2005). This suggested that Tay primary function is to prevent increases in EGFR/Erk signalling in places where the pathway must be active but only at low levels. Thus, high levels of EGFR activity and dP-Erk accumulation are restricted to the presumptive veins in wild type third instar wing discs, but the pathway is also active at lower levels in the inter-veins, where it promotes cell proliferation and survival. In tay or Mkp3 mutant backgrounds, a fraction of these cells initiates the vein differentiation program, escaping the negative feed-back loops that maintain low dP-Erk levels and entering the positive feed-back loops that normally operate in vein territories through the regulation of rhomboid expression. In this model, Tay would participate in a mechanism that favours Erk de-phosphorylation and its nuclear retention in an inactive form. This mechanism of Tay action is compatible with the effects of its over-expression, which essentially cause a failure to accumulate dP-Erk in vein territories, and consequently a loss of vein differentiation (Molnar, 2013).

Signalling by Erk proteins in the nucleus is in part regulated by the rate of Erk nucleus/cytoplasm shuttling. In the nucleus, signal termination involves Erk de-phosphorylation by nuclear phosphatases and also its sequestration away from cytoplasmic Erk kinases. Because Erk does not contain nuclear localization nor export sequences, its subcellular localization relies on proteins acting as anchors. This study has observed direct interactions between Tay and Erk and between Tay and Mkp3, and these interactions were also detected in immunoprecipitation experiments from embryo protein extracts. These data suggests that Tay could form part of protein complexes including both Erk and Mkp3 in the nucleus (Molnar, 2013).

A direct interaction between Tay and Erk is also compatible with several observations regarding Tay stability and Erk subcellular localization. First, Erk and Erksem increase the accumulation of Tay in the nucleus, and do so independently of EGFR signalling, as neither RasV12 nor Mkp3 over-expression modified Tay accumulation. Second, Tay over-expression prevents the accumulation of dP-Erk, whereas loss of Tay has the converse effect. Finally, Tay over-expression modifies Erksem subcellular localization, increasing the nucleus/cytoplasm ratio of Erksem accumulation. In this regard, it is worth noting that the expression of RasV12 has the same effects on Erksem subcellular localization as the over-expression of Tay, as both Tay and RasV12 increase the nuclear/cytoplasm ratio of Erksem accumulation. It was noticed that the effects of Tay on Erk localization are only manifest when the Erksem form was used. Because it was also seen that Erksem is not retained in the cytoplasm by Mkp3, it was reasoned that Erksem, liberated of cytoplasmic anchorage by Mkp3, is more sensitive to pathway activation and to the presence of other anchoring proteins, and that Tay might play this role in the nucleus (Molnar, 2013).

A direct interaction was observed between Tay and Mkp3. Mkp3 is a dual-specificity phosphatase that is predominantly localised in the cytoplasm, but it shuttles between the nucleus and cytoplasm and could play a role in translocating inactive Erk from the nucleus to the cytoplasm. It is possible that Tay could promote the nuclear function of Mkp3, but in addition, Tay should also act independently of Mkp3 to promote Erk inactivation and retention, because Tay is able to down-regulate Erk activity in Mkp3 mutant backgrounds (Molnar, 2013).

Most of the Tay interacting region with Erk is localised to the C-terminal part of Tay, a 1000 amino acid long region that includes the domain of homology between Tay and human AUTS2. This fragment of Tay fails to interact with Mkp3, and is even more efficient than the full-length protein in its effects on Erk subcellular localization and in its antagonism on Erk signalling. Intriguingly, AUTS2 expressed in the wing disc also interferes with EGFR signalling, but it does so in an opposite manner to Tay or to the Tay C-terminal domain. Few conclusions can be extracted from the consequences of AUTS2 expression in the wing disc, but it is speculated that this protein retains some of its interactions with Drosophila Erk that might protect this protein from inactivation by nuclear phosphatases. Similarly, the effects of AUTS2 on Drosophila EGFR signalling are compatible with a role for this protein in the regulation of Erk activity in humans, and that this effects might underline the effects of zebrafish, murine and human mutations in the onset of neurological disorders (Molnar, 2013).

From the analysis in the wing disc it is concluded that Tay interacts with Erk in the nucleus, affecting its phosphorylation and promoting its nuclear retention. In this context, it is interesting to note that the free diffusion of human ERK2 is impeded within the nucleus, and that this limitation in mobility increases after ERK2 stimulation. This has lead to postulate that ERK2 retention in the nucleus involves high-affinity interactions with unidentified low-mobility sites that are constitutively expressed. This study suggests that Tay could play such a role in vivo, acting as a nuclear anchor for Erk that facilitates its inactivation by nuclear phosphatases and its retention in an inactive state (Molnar, 2013).

Nutrient-dependent requirement for SOD1 in lifespan extension by protein restriction in Drosophila melanogaster

Diet composition is a critical determinant of lifespan, and nutrient imbalance is detrimental to health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. This study investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extends lifespan in females fed low carbohydrate-to-protein (C:P) diets but not the high C:P ratio diet. This extension is associated with increased resistance to oxidative stress; transcriptional changes in metabolism, proteostasis, and immune genes; reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and mitogen-activated protein kinase (MAPK) signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. These data reveal a link among diet, mitochondria, and MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging (Sun, 2014).

Dietary nutrients are among the most critical environmental factors that modulate healthspan and lifespan. Nutrient imbalance is a major risk factor to human health and common among old people. Dietary restriction (DR), by reducing the amount of all or specific nutrients, is a potent nongenetic intervention that promotes longevity in many species. In general, protein restriction is more effective in influencing lifespan than sugar or calorie restriction in Drosophila. However, increasing evidence indicates that the composition of dietary nutrients, such as carbohydrate-to-protein (C:P) ratio, is more critical than individual nutrients in affecting health and lifespan. Optimal lifespan peaks at the C:P ratio 16:1 in Drosophila and 9:1 in Mexican fruit fly. A recent study in mice shows that lifespan is primarily regulated by the C:P ratio in the diet and tends to be longer with higher C:P ratios. Diet composition is also critical for DR to promote longevity in nonhuman primate rhesus monkeys. Two major nutrient-sensing pathways are known to modulate lifespan. One is target-of-rapamycin (TOR) signaling that mostly senses cellular amino acid content and the other is insulin/insulin-like signaling that primarily responds to circulating glucose and energy levels. Excessive carbohydrate and protein intake both contribute to development of insulin resistance and diabetes in animal models and humans. Dietary macronutrients, such as sugar, protein, and fat, may interact with each other to influence nutrient-sensing pathways and consequently health outcome. It is thus critical to take into account diet composition in elucidating molecular mechanisms of aging and in developing effective interventions for promoting healthy aging (Sun, 2014).

Aging is associated with transcriptional and translational changes in many genes and proteins. Some age-related changes are evolutionarily conserved, and many function in nutrient metabolism, such as mitochondrial electron transfer chain (ETC) genes, many of which are downregulated with age in worms, flies, rodents, and humans. Knocking down ETC genes affects lifespan in yeast, worms, and flies. Mitochondrial genes also play a key role in numerous age-related diseases, such as Parkinson's and Alzheimer's disease. However, how mitochondrial genes interact with nutrients to modulate lifespan and health-span remains incompletely elucidated. Understanding gene-environment interactions will be a key to tackle aging and age-related diseases (Sun, 2014).

ATP synthase subunit d (ATPsyn-d) is a component of ATP synthase, ETC complex V, and is known to modulate lifespan in C. elegans. How ATPsyn-d modulates lifespan and whether it functions in modulating lifespan in other species remain to be determined (Sun, 2014).

Given the importance of nutrients as environmental factors in modulating lifespan, this study has investigated whether and how ATPsyn-d interacts with dietary macronutrients to modulate lifespan in Drosophila. ATPsyn-d was found to interact with dietary macronutrients to influence accumulation of oxidative damage and protein aggregates; resistance to oxidative stress; and expression of numerous genes involved in metabolism, proteolysis, and innate immune response and more importantly to modulate lifespan. Moreover, ATPsyn-d affects mitogen-activated protein (MAP) kinase (MAPK) signaling and genetically interacts with TOR signaling to influence lifespan of flies in a diet-composition-dependent manner. This study reveals the critical interaction between mitochondrial genes and nutritional factors and the underlying mechanisms involving TOR signaling in modulating lifespan (Sun, 2014).

Considering the essential role of mitochondrial function in metabolism and aging, this study investigated how diet composition influences the function of ATPsynd, a component of mitochondrial ATP synthase, in aging and the underlying mechanisms. ATPsyn-d knockdown extends lifespan in Drosophila under low sugar-high protein diets, but not under a high sugar-low protein diet. Lifespan extension induced by ATPsyn-d knockdown is associated with increased resistance to oxidative stress and improved protein homeostasis. Furthermore, evidence is provided suggesting ATPsyn-d modulates lifespan through genetically interacting with TOR signaling. Knocking down of atp-5, the worm ATPsynd, extends lifespan in C. elegans, along with the current data suggesting a conserved role of ATPsyn-d in modulating lifespan. Altogether, these findings reveal a connection among diet, mitochondrial ATP synthase, and MAPK and TOR signaling in modulating lifespan and shed light on the molecular mechanisms underlying the impact of diet composition on lifespan (Sun, 2014).

The following model is proposed to explain how ATPsyn-d interacts with dietary macronutrients to modulate lifespan, considering the genetic interaction between ATPsyn-d and TOR signaling and the fact that suppression of TOR signaling by altering expression of its components, such as Tsc1/Tsc2, S6K, and 4E-BP, activates autophagy, improves proteostasis, and promotes longevity in high-protein diets, but not necessarily low-protein diets. It is postulated that TOR signaling is regulated by ATPsyn-d and perhaps other mitochondrial proteins. ATPsyn-d knockdown reduces MAPK signaling and probably affects other signaling pathways, which may consequently decrease TOR signaling to extend lifespan in Drosophila fed high-protein diets, such as SY1:9 and SY1:1, but not low protein diets. It is possible that diet-dependent response is due to knockdown of ATPsyn-d protein to different extent by RNAi under different dietary conditions. This is not likely the case. The amount of ATPsyn-d knockdown is not much different between flies on sugar (S) and yeast (Y) SY1:9 and SY9:1, although lifespan is not increased by ATPsyn-d knockdown for flies under SY9:1. Therefore, variations in ATPsyn-d knockdown under current experimental conditions unlikely contribute significantly to diet-dependent lifespan extension. Consistent with this model, ATPsyn-d knockdown increases resistance to acute oxidative stress, reduces cellular oxidative damage, and improves proteostasis in Drosophila. Reduced oxidative damage by ATPsynd knockdown may lead to decreased MAPK signaling, which in turn modulates TOR signaling and proteostasis (Sun, 2014).

Another likely scenario would be that ATPsyn-d and TOR signaling form a positive but vicious feedback loop through MAPK signaling to induce molecular, metabolic, and physiological changes detrimental to lifespan. This vicious cycle can be disrupted by high-C:P diet, knockdown of mitochondrial genes, or suppression of TOR signaling pharmacologically by rapamycin or genetically by Tsc2 overexpression. Consistent with this possibility is that ATPsyn-d knockdown reduces phosphorylation of S6K, a component of TOR signaling, and increases expression of genes involved in maintaining proteostasis and possibly autophagy, which are regulated by TOR signaling. The level of pS6K reflects the strength of TOR signaling, and reduction- of-function mutants of S6K are known to extend lifespan in several species. ATPsyn-d may genetically interact with TOR signaling to modulate lifespan by influencing protein levels of both S6K and pS6K, although it does not necessarily affect the pS6K/S6K ratio, which may not be a reliable indicator for the strength of TOR signaling under the three SY diets due to the change of S6K level. Furthermore, ATPsyn-d knockdown reduces oxidative damage and polyubiquitinated protein aggregates, which are biomarkers of aging. Rapamycin reduces lifespan extension induced by ATPsyn-d knockdown, which may be due to exacerbation of some detrimental effects of reduced TOR signaling. However, this observation further supports the connection between ATPsyn-d and TOR signaling. Although both rapamycin and ATPsyn-d knockdown reduce pS6K level, ATPsyn-d knockdown, but not rapamycin, decreases S6K level, suggesting ATPsyn-d knockdown and rapamycin affect TOR signaling in different manners. Further studies are warranted to clarify the epistatic relationship between ATPsyn-d and TOR signaling (Sun, 2014).

Increasing evidence has demonstrated the importance of diet composition or carbohydrate to protein ratio in modulating lifespan and health. Nutrient geometry studies conducted in Drosophila have shown that C:P ratio in the diet is far more important in determining lifespan than calorie content or single macronutrient. A recent tour de force nutrient geometry study in mice has confirmed and expanded the view on the critical role of C:P ratio in regulating lifespan and cardiometabolic health to mammals. An important implication from nutrient geometric studies is that diet composition would have a significant impact on the effectiveness of inventions for promoting healthy aging by genetic, pharmaceutical, or nutraceutical approaches (Sun, 2014).

This indeed is the case, although evidence comes from only a handful of studies. Rapamycin feeding extends lifespan in yeast, worms, flies, and mice. Although rapamycin feeding has been shown to extend lifespan of flies under a broad range of diets, some studies have shown that rapamycin feeding does not extend lifespan in flies under high carbohydrate-low protein diets. Supplementation of a nutraceutical derived from cranberry extends lifespan in female flies under a high-C:P-ratio diet, but not a low-C:P-ratio diet. Suppression of TOR signaling by overexpression of Tsc1/Tsc2 extends lifespan in flies under relatively higher-protein diets, but not under low-protein diets, although those studies focused on the variation of protein concentration instead of C:P ratio. Consistent with the link between ATPsyn-d and TOR signaling, ATPsyn-d knockdown extends lifespan in female flies under low sugar-high protein diets, but not high sugar-low protein diet, likely due to the fact that TOR signaling is already low under the high sugar-low protein diet. It was further shown that rapamycin feeding extends lifespan in wild-type female flies, but not in ATPsyn-d knockdown flies (Sun, 2014).

Aging is associated with profound decline in protein homeostasis, and many longevity-related pathways, such as TOR and insulin-like signaling, modulate lifespan through improving proteostasis. Suppression of TOR signaling extends lifespan through decreasing protein translation and increasing autophagy, key processes for maintaining proteostasis. This study found that ATPsyn-d knockdown reduces the level of 4-HNE (an α, β-unsaturated hydroxyalkenal that is produced by lipid peroxidation) protein adducts; a biomarker for lipid protein oxidation; and the level and aggregation of polyubiquitinated protein, a biomarker for proteostasis and aging. ATPsyn-d is a key component of mitochondrial ATP synthase complex. Along with the link between ATPsyn-d and TOR signaling, these data suggest that mitochondrial ATP synthase is critical for maintaining proteostasis and modulating lifespan. This notion is further supported by a recent study showing that α-ketoglutarate, an intermediate in the TCA cycle, suppresses mitochondrial ATP synthase probably by binding to ATP synthase subunit b (ATPsyn-b) and also inhibits TOR signaling to extend lifespan in C. elegans. However, it remains to be determined whether suppression of ATP synthase by α-ketoglutarate results in inhibition of TOR signaling in C. elegans or any other species. It is also likely that ATPsyn-d and ATPsyn-b influences ATP synthase and TOR signaling through different mechanisms, because α-ketoglutarate reduces cellular ATP level in C. elegans, whereas ATPsyn-d knockdown does not significantly change or even increase ATP level in Drosophila. This also suggests that lifespan extension is not necessarily associated with decreased ATP level, which is supported by a study in Drosophila showing that any change of ATP level is not correlated with any change of lifespan induced by knockdown of a number of mitochondrial genes. Nevertheless, these studies suggest that ATP synthase is a key and conserved player linking dietary nutrients from TOR signaling to proteostasis and lifespan (Sun, 2014).

Similar to many longevity-related mutants, lifespan extension induced by ATPsyn-d knockdown is associated with reduced oxidative damage and increased resistance to oxidative stress. ATPsyn-d knockdown increases lifespan and resistance to paraquat, an acute oxidative stress response, under SY1:9 or SY1:1. However, ATPsyn-d knockdown increases resistance to paraquat but does not extend lifespan in female flies under SY9:1. In addition, ATPsyn-d knockdown decreases 4-HNE level, an indicator of accumulated oxidative damage, under SY1:9, but not SY1:1. These indicate that the effect of ATPsyn-d knockdown on oxidative damage and lifespan depends on diet composition, suggesting that oxidative stress resistance is at most partially responsible for lifespan extension. This should not be surprising because it is consistent with numerous studies in the literature showing that stress resistance does not always result in lifespan extension despite the strong link between oxidative stress and aging (Sun, 2014).

The role of mitochondrial genes in modulating lifespan is complex. Knockdown of some electron transfer chain (ETC) genes increases lifespan whereas knockdown of others decreases or does not alter lifespan in C. elegans and Drosophila. This study reveals another layer of complexity regarding the role of ETC genes in lifespan modulation, namely the impact of diet composition. These findings indicate that ATPsyn-d knockdown promotes longevity at least partially through TOR signaling. TOR signaling senses cellular amino acid content and regulates numerous biological processes, including translation, autophagy, and lifespan. 4E-BP, a translational repressor in TOR signaling, mediates lifespan extension induced by dietary restriction (Sun, 2014).

Activated 4E-BP suppresses general translation but selectively increases translation of some mitochondrial ETC genes, the latter of which results in increased mitochondrial biogenesis and potentially lifespan. Lifespan extension induced by dietary restriction is suppressed by knocking down ETC genes regulated by 4E-BP. The findings by Zid suggest that increased protein expression of some ETC genes is associated with lifespan extension induced by dietary restriction. However, unlike those ETC genes, ATPsyn-d knockdown extends instead of decreases lifespan under high-protein diets. Therefore, it is likely that ETC genes can be categorized into two groups: one selectively upregulated by activated 4E-BP and the other insensitive to activated 4E-BP, the latter of which may include ATPsyn-d. The two groups of ETC genes may interact with dietary macronutrients to modulate lifespan perhaps through different modes of action. Future studies are warranted to investigate the dichotomous role of translation of ETC genes in modulating lifespan (Sun, 2014).

Downstream Targets of Rolled

Continued: MAP kinase Protein Interactions part 2/3 | part 3/3

rolled/MAPK: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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