Effects of Mutation or Deletion

A salient feature of these findings is that equivalence groups form in response to the localized functions of two RTKs acting within the larger prepatterned region. The mesodermal diphospho-MAPK expression pattern not only provides strong support for this hypothesis, but also raises the question of how the upstream receptors are activated in discrete subsets of competent cells. For C15, this may occur through induction by P2, which expresses Rhomboid, a factor that can nonautonomously stimulate Egfr in adjacent cells. The localized activation of Htl could result from restricted expression of its as yet unidentified ligand. Htl itself is enriched in groups of Eve-positive mesodermal cells, a process that might also contribute to the Htl-dependent formation of these clusters. RTK signaling in the Drosophila embryonic mesoderm is important for cell migration and cell fate specification. The latter role can be divided into several distinct functions: (1) Htl and DER promote the formation of L'sc clusters or equivalence groups; (2) Ras1 signaling activates identity gene expression in the entire group of equivalent cells; (3) activated MAPK becomes restricted to progenitors, suggesting a role for RTK signaling in progenitor selection, and (4) MAPK is reactivated in one of the sibling founders derived from a single progenitor, suggesting that RTK activity helps to establish or maintain the founder identity that is initiated by the asymmetric division of its progenitor. Alternatively, RTK function in some founder cells could promote their differentiation. The ability of activated Ras1 to generate supernumerary Eve founders in mbc mutant embryos is consistent with this last possibility. Interestingly, the RTK/Ras1 pathway is similarly utilized in a sequential manner during development of the Drosophila compound eye (Carmena, 1998 and references).

In the Drosophila eye, activation of the sevenless (sev) receptor tyrosine kinase is required for the specification of the R7 photoreceptor cell fate. In a genetic screen for mutations that result in the activation of the sev signaling pathway in the absence of the inducing signal, a gain-of-function mutation in rolled (rlSevenmaker [rlSem]) has been identified, which encodes a homolog of mitogen-activated protein (MAP) kinase. In addition to the sev pathway, this mutation also activates the pathways controlled by Torso and the Epidermal growth factor receptor. The rlSem mutation results in the substitution of a single conserved amino acid in the kinase domain. Activation of MAP kinase by the rlSem mutation is both necessary and sufficient to activate multiple signaling pathways controlled by receptor tyrosine kinases (Brunner, 1994a).

Three lines of evidence establish that rolled encodes a Drosophila MAP kinase required in the Sevenless signal transduction pathway: 1) rl alleles are dosage-sensitive suppressors of a constitutively activated form of raf expressed in the R7 equivalence group. Constitutively active raf produces a rough eye phenotype. Reduction in rl activity also reverses the suppression of sevenless by Sos mutants. These results indicate that rolled acts downstream of raf in the Sevenless signaling pathway. 2) Molecular analysis reveals that two alleles of the rolled locus contain mutations leading to internally deleted and C-terminally truncated forms of ERK-A. 3) Expression of the ERK-A cDNA complement the larval lethally of rolled null alleles, as well as reversing the effects of a reduction in rolled gene dosage during eye development (Biggs, 1994).

The small wing (sl) gene encodes a Phospholipase C-gamma (PLC-gamma) involved in eye and wing morphogenesis and is also likely to play other essential roles in fly biology. To determine whether there are interactions between sl and other genes of the Ras pathway, a mutation of MAP kinase, rolled (rl1) was used. This mutation is viable and thus its effects can easily be examined in the adult eye (homozygous loss-of-function mutations of most known components acting downstream of the Sev and Egfr RTKs are embryonic lethal). The partial loss-of-function mutation of MAPK, rl1, has a mild impact on R7 formation: 22% of the ommatidia lack R7 cells. Both sl;rl1 double mutants have phenotypes comparable to the rl1 single mutant: 36% and 27%, respectively, are missing R7 cells and <1% contain extra R7 cells. Thus, whereas in sl mutants 51% of ommatidia have extra R7 cells, this is reduced to <1% in a rl1 background. The phenotype of the sl;rl double mutants suggests that sl is acting upstream of rl. To confirm this result, interaction was tested between sl and a gene downstream of rl, seven in absentia (sina), which encodes a nuclear protein and for which a viable loss-of-function allele, sina2, is also available. In sina2, the proportion of ommatidia with an R7 cell is less than 5% . Adding either an sl mutation into a sina2 background causes no increase in the proportion of ommatidia with R7 cells. Thus the results with sina are consistent with those from rl, indicating that sl affects Ras signaling upstream from the rl MAP kinase. The wing length phenotype observed in the sl single mutants is unaffected in any of the homozygous double mutant combinations. In contrast, the ectopic wing vein phenotypes of sl mutants are strongly suppressed by rl1 : in sl, 49% of wings (n=43) have ectopic veins, compared to only 7% in the double mutant sl;rl 1 (Thackeray, 1998).

Drk, a Drosophila SH2 adaptor protein and Sos, a putative activator of Ras1, Ras1, raf and Rolled/MAP kinase have all been shown to be required for signaling from the Sevenless and the Torso receptor tyrosine kinase. From these studies, it is unclear whether SOS and its targets act in a single linear pathway as suggested by the genetic analysis or whether different components serve to integrate different signals. Removing each of these components during the development of the adult epidermal structures produce a very similar set of phenotypes. These phenotypes resemble those caused by loss-of-function mutations in the Drosophila EGF receptor homolog (DER). It appears that these components form a signaling cassette, which mediates all aspects of DER signaling but that is not required for other signaling processes during epidermal development (Diaz-Benjumea, 1994).

vein and spitz show a strong genetic interaction suggesting a molecular interdependence. Reducing vein dose in a spi null genotype dramatically worsens the phenotype to produce a collapse embryo with an extruded head skeleton. However, the double mutants are not as severly affected as are Egf-R null mutants. Genetic interactions are also observed between vn, Egf-R and rolled. The gain of function alleles Egf-R-Ellipse and rolled-Sevenmaker rescue proliferation defects in strong and null vein mutants. These defects include a small wing disc and the size of the pupal case (Schnepp, 1996).

The Drosophila Ras1 gene is required for proper cell fate specification throughout development; the loss-of-function phenotype of Ras1 suggests an additional role in cell proliferation or survival. A direct role for Ras1 in promoting cell proliferation, however, has not been established. Expression of an activated form of Ras1 (Ras1[V12]) during Drosophila wing imaginal disc development is sufficient to drive ectopic cell proliferation and hyperplastic tissue growth. Expression of Ras1(V12) induces widespread cell death in the imaginal discs, including cells not expressing the transgene, which results in ablation of adult structures. It is thought that the non-autonomous cell death induced by ectopic Ras could be a manifestation of cell competition Increased cell death may represent a mechanism to compensate for excessive proliferation and regulate the overall disc size. Loss-of-function mutations in the genes encoding RAF, MEK, MAPK and KSR dominantly suppress Ras1(V12)-induced cell proliferation. Two Ras effector loop mutations (E37G and Y40C) that block the Ras-RAF interaction, also suppress Ras1(V12)-induced proliferation, consistent with a requirement for the MAPK cascade during the Ras1 mitogenic response. These two Ras effector loop mutants, however, retain some activity and can act synergistically with a MAPK gain-of-function mutation, suggesting that Ras1 may also act through signaling pathway(s) distinct from the MAPK cascade, but which act in parallel with the MAPK pathway (Karim, 1998).

Mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. Double mutant combinations were studied between mutations affecting the activity of rolled /MAP kinase and several genes that are essential for the establishment of a bipolar spindle during progression through mitosis; no interactions with mutations in polo, mgr, or aurora were found. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors (Inoue, 1998).

To investigate a Ras-independent means of activating the Mapk cascade, mutations have been isolated that suppress the lethality of a Drosophila Raf mutation [also referred to as l(1) pole hole]. Six extragenic Su(Raf) loci have also been identified. These mutations not only suppress RafC110 but also other partial loss-of-function Raf alleles that do not impair Ras-Raf binding. This suggests that the suppression of RafC110 by the extragenic Su(Raf) mutations does not necessarily involve the restoration of Ras-Raf binding. Developmental analyses have shown that all six extragenic Su(Raf) mutations promote signaling in the Sevenless (Sev) and Egfr RTK pathways. Su(Raf)34B is a gain-of-function mutation in the Dsor1 locus that encodes the fly Mek. Recently, Su(Raf)1 has been shown to encode Src42A. The isolation of mutations that suppress the suppressor activity of Su(Raf)1 is reported in this paper. These mutations define two known genes, Egfr and rolled (rl; also referred to as Mapk) and two previously uncharacterized loci. In addition, two alleles of Src42A were also isolated in the screen, although these mutations are not true suppressors of Su(Raf)1 (Zhang, 1999).

One of the novel suppressor loci was named semang (sag). sag is required during both embryonic and imaginal disc development. Mutations in sag cause zygotic lethality. To identify developmental pathways where sag functions, the phenotypes associated with sag mutations were examined with particular attention to those processes controlled by known Drosophila RTKs. The results of these analyses show that sag participates in the Torso (Tor) and Drosophila DFGF-R1 RTK (Breathless) pathways during embryonic development. sag also disrupts the embryonic peripheral nervous system. During imaginal disc development, sag mutations affect two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions broadly in several RTK-mediated processes. This role of sag in RTK signaling is further supported by the genetic interaction between sag and other known RTK signaling genes. sag dominantly enhances the phenotypes caused by reductions of RTK signaling in loss-of-function Raf or rl mutants. Consistent with this, sag dominantly suppresses the formation of supernumerary R7 cells caused by the activated sev-Ras1V12 mutation. The sag mutations analyzed are likely to be loss-of-function mutations. These results suggest that sag may have a positive role in RTK signaling (Zhang, 1999).

Drosophila has two other Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. Src64 does not affect viability when mutated. The isolation of Su(Raf)1 as a mutation in Src42A that restores the viability of Raf mutants and the isolation of Egfr, rl, and sag as extragenic suppressors of Su(Raf)1 provides the first in vivo evidence that both Src42A and sag are modulators of RTK signaling. At this moment, it is not known where Src42A and sag fit into the known RTK signaling cascade. An Src42A cDNA driven by a ubiquitously expressing promoter rescues the lethality of both Su(Raf)1 homozygotes and Su(Raf)1/Df hemizygotes. Based on this, Su(Raf)1 has loss-of-function characteristics, suggesting that Src42A is, unexpectedly, a negative modulator of RTK signaling. However, the genetics of Su(Raf)1 suggest that the suppression of RafC110 may be attributed to a dominant-interfering effect because the RafC110 lethality is not suppressed in Src42A hemizygotes of genotype Df(2R)nap9/+. Because of this, the role of Src42A in RTK signaling is still being investigated. However, the genetic interaction as revealed by the modifying screen suggests that Egfr and other RTKs may possibly regulate Src42A and sag, which in turn modulate the Mapk cascade (Zhang, 1999).

Genetic and molecular characterization of the dominant suppressors of D-rafC110 on the second chromosome have identified two gain-of-function alleles of rolled (rl); rl encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rlSu23, was found to bear the same molecular lesion as rlSem, which has been reported to be dominant female sterile. However, rlSu23 and the current stock of rlSem show only a weak dominant female sterility. This discrepancy could be due to a difference in the genetic background: either the presence of a mutation(s) that enhances Tor signaling in the original rlSem stock or the occurrence of a suppressor mutation(s) in the current stocks of both rlSem and rlSu23. So far, outcrossings of the current stocks have shown no evidence of suppressor mutations. Detailed analyses of the rl mutations demonstrate moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations fail to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation has also been demonstrated by clonal analysis (Lim, 1999).

It has been reported that the increased signal sensitivity of the mammalian ERK2D319N protein that has a mutation analogous to RlSem is due to a decreased sensitivity to dual-specificity MAPK phosphatases such as PAC1, CL100/MKP-1, MKP-2, and MKP-3, rather than to an increased kinase activity. However, an in vitro kinase assay of the recombinant RlSem mutant protein produced in bacteria demonstrates significant activity for the phosphorylation of Yan, a native substrate of Rl, in the absence of activating MAPKK, while the normal recombinant Rl does not. In the presence of activated mammalian MAPKK, RlSem exhibits a higher kinase activity than Rl+. The latter observations suggest an increased basal level activity of RlSem in addition to an increased sensitivity to the activator. The constitutive activity observed in vitro is consistent with the significant suppressor activity of rlSu23 in the proliferation defect in the null Dsor1Gp158 clones. Taking these observations into account, it is most likely that the dominant activity of the RlSem and RlSu23 mutant proteins is due to both an increased basal level activity and a decreased sensitivity to inactivating phosphatases (Lim, 1999).

On the basis of the above considerations, it is proposed that the activation of Rl is necessary but not sufficient for Tor signaling, and that Dsor1 may provide yet another branching point in the Tor signaling pathway. One possible model would be that Dsor1 activates another unknown factor in addition to Rl in the Tor pathway, and that both are required for the transcriptional activation of tll and hkb. It would also be possible that an inactivation of a factor that antagonizes the Rl function by Dsor1 would be required for the activation of the pathway. Defects of varying degrees were seen in mitoses in the syncytial blastoderm embryos devoid of the maternal Dsor1 activity, suggesting that Dsor1 participates in the regulation of mitosis and is activated throughout the embryo during cleavage divisions. Bifurcation of the Tor signals downstream of Dsor1 may constitute a mechanism for preventing Dsor1 from activating the target genes in regions other than the terminal regions of the embryo. Integration of signals for imaginal cell proliferation would then take place at some other point in the MAPK cascade. The differential branching and integration of signals may contribute to the functional diversification of the ubiquitous MAPK cascade (Lim, 1999).

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, several specific requirements have been identified for da+ in somatic cells during follicle formation. (1) da is a critical regulator of somatic cell proliferation. (2) da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. (3) da is a genetic regulator of an early checkpoint for germline cyst progression: loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles (Smith, 2002).

Although da's role in the control of somatic proliferation is unknown, it probably involves regulation of cell cycle progression. Connections between EGFR signaling and cell cycle progression have already been established in the R2-R5 photoreceptor cells in the morphogenetic furrow of the eye, where EGFR signal transduction is required for G2/M progression, but signal inactivity is necessary for G1/S progression. Coincidentally, Da protein levels are high in those cells, and da function is required for their G1/S progression. A similar connection between da and cell cycle control in the ovary is implicated by the observation that da exhibits genetic interaction phenotypes with both loss of function (rl-) and persistently activated (rlSem) MAPK alleles. Loss of EGFR signaling would be expected to delay cell cycle progression at the G2/M transition, but persistent MAPKSem activity, being a poor substrate for the inactivating phosphatase, would delay the cell cycle at the G1/S transition. In either situation additionally reducing the da dose (which itself would slow G1/S progression) would lead to the mutant phenotype observed in genetic interactions, and the higher frequency of defects with rlSem is consistent with both da and rlSem impacting on the same stage of the cell cycle (Smith, 2002).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Rolled is a mitogen-activated protein kinase that functions at the last step in the Ras-MAPK phosphorylation cascade. Activated Rolled activates downstream transcription factors and thus plays a key role in the Egfr-mediated signaling required for cell determination and pattern formation. rll/+; rg/Y double mutants were constructed to test for genetic interactions with rg, and a single copy of the rolled loss-of-function mutation enhances the rough eye phenotype of rg. A single copy of the dominant gain-of-function rolled mutation acts a suppressor of the rough eye phenotype. Taken together these results suggest that rugose interacts with the components of the signal cascade activated by Egfr (Shamloula, 2002).

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, transgenic fruitflies were generated with GAL4-inducible expression of wild type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles was lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele causes ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses have established that the cswN308D ectopic wing vein phenotype requires intact EGF ligand and receptor, and that this transgene interacts genetically with Notch, DPP and JAK/STAT signaling. LOF alleles of positive regulators (downstream of receptor kinase, son of sevenless, Ras85D, Downstream of raf1, rolled, pointed, Hsp83) resulted in statistically significant suppression of ectopic vein formation. Most wings showed no or minimal ectopic vein formation in the anterior part of the wing, which were consistently observed in the UAS-cswN308D/+;tub-GAL4/+ control wings. In contrast, LOF alleles of negative regulators (sprouty, Gap1) enhanced the phenotype with longer ectopic veins in the anterior part of the wings andmultiple and complex ectopic vein formation in the posterior part. LOF alleles of Egfr, its ligand (vein) and positive extracellular regulators (Star, rhomboid) suppressed the wing phenotype while LOF alleles for argos, a negative ligand regulator, enhanced it. Expression of the mutant csw transgenes increases RAS-MAP kinase activation, which is necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers (Oishi, 2006).

Rolled regulates synaptic plasticity at the neuromuscular junction

Ras proteins are small GTPases with well known functions in cell proliferation and differentiation. In these processes, they play key roles as molecular switches that can trigger distinct signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide-3 kinase pathway, and the Ral-guanine nucleotide dissociation stimulator pathway. Several studies have implicated Ras proteins in the development and function of synapses, but the molecular mechanisms for this regulation are poorly understood. The Ras-MAPK pathway is involved in synaptic plasticity at the Drosophila larval neuromuscular junction. Both Ras1 and MAPK are expressed at the neuromuscular junction, and modification of their activity levels results in an altered number of synaptic boutons. Gain- or loss-of-function mutations in Ras1 and MAPK reveal that regulation of synapse structure by this signal transduction pathway is dependent on Fasciclin II localization at synaptic boutons. These results provide evidence for a Ras-dependent signaling cascade that regulates Fasciclin II-mediated cell adhesion at synaptic terminals during synapse growth (Koh, 2002).

Synapse stability and synapse expansion during muscle growth are regulated by changes in FasII expression at presynaptic and postsynaptic membranes and FasII expression is in part controlled by electrical activity. One mechanism through which electrical activity alters FasII levels is by regulating its synaptic clustering via CaMKII-dependent phosphorylation of Discs large. An additional mechanism by which the levels of FasII at the presynaptic terminal are modified has been documented in this study: the activation of the Ras-MAPK pathway. This redundant mechanism may serve the differential regulation of FasII localization at the presynaptic and postsynaptic site or may represent FasII regulation in response to different signals. Whereas activation of CaMKII is elicited by an increase in electrical activity, activation of the MAPK pathway may be triggered by activity or by an as yet unknown but different signaling mechanism (Koh, 2002).

Studies in Aplysia indicate that activity-dependent endocytosis of ApCAM results in an increase in the number of synaptic contacts during long-term facilitation. ApMAPK is likely to induce ApCAM internalization in a process that depends on ApMAPK activity in dissociated neurons. However, its involvement in the intact organism has not been tested (Koh, 2002 and references therein).

In this study, Drosophila larval neuromuscular synapses have been used to determine the involvement of the Ras-MAPK pathway in the regulation of synaptic FasII levels and in morphological synaptic plasticity. Both Ras and MAPK are expressed at the NMJ, where they regulate presynaptic expansion. This regulation is accomplished by altering FasII levels at synaptic boutons. A ras hypomorph mutant and anti-Ras antibodies have been used to determine that Ras1 is specifically expressed at the larval NMJ. Although Ras1 immunoreactivity at synapses and muscles is severely reduced in ras1 hypomorphic mutants, nuclear staining persists (Koh, 2002).

Two antibodies were used to demonstrate the synaptic localization of MAPK at the NMJ, an antibody that recognizes all forms of the MAPK Rolled (DmERK-A) and an antibody that exclusively labels active, double-phosphorylated MAPK (DpMAPK). Interestingly, although both antibodies labeled synaptic boutons, their distribution was not identical. In particular, the antibody against active MAPK-labeled hot spots was more restricted in its localization than general MAPK staining. This suggests that active MAPK is recruited to specific domains within the synaptic bouton or that MAPK activation occurs at discrete regions within the boutons. Interestingly, the same domain that is occupied by active MAPK has lower levels of FasII, consistent with the idea that MAPK activation might be involved in the downregulation of FasII. It has been suggested that the regions of low FasII concentration correspond to the active zone, suggesting that active MAPK is localized to the active zone. The localization pattern of Ras1 and MAPK at synapses is also consistent with the localization protein 14-3-3, another protein that has been involved in the Ras1-Drosophila Raf-MAPK signal transduction pathway (Koh, 2002).

Expression of constitutively active Ras (Ras1V12) drastically increases the number of synaptic boutons. This change is indistinguishable from the increase in boutons observed in the Ras1V12S35 variant and the constitutively activated RafF179, suggesting that these changes are induced by activation of the MAPK pathway. Consistent with these results is the observation that a hypomorphic mutation in ras1, ras15703, has the opposite phenotype, a decrease in bouton number, and that a gain-of-function mutation in rl leads to an increase in bouton number. The finding that Ras1V12 and Ras1V12S35 elicit identical phenotypes at the NMJ is consistent with findings in other tissues, such as in the retina, in which the epidermal growth factor receptor-Ras1 pathway is involved in photoreceptor survival, or in the wing discs, where the Ras pathway is involved in hyperplastic growth (Koh, 2002).

Notably, expression of Ras variants that activate the PI3-K and Ral signal transduction pathways and a constitutively active RalA also induce an increase in bouton number that is similar in extent to RasWT and considerably lower than Ras1V12. These results raise the possibility that Ras1V12G37 and Ras1V12C40 may still retain some degree of affinity for Raf or, alternatively, that other Ras-mediated pathways might also influence NMJ development. All known ras genes encode a protein region, the effector loop, that is highly conserved in all species. Mutations in this loop interfere with the ability of Ras to bind to specific effectors without altering its catalytic activity. A series of mutations in the effector loop that allow almost exclusive activation of a single effector havs been isolated in mammals. The specificity of these mutants has been tested by in vitro binding assays as well as by genetic and biochemical approaches in cell culture. In Drosophila, a genetic approach has been used to demonstrate specificity. These studies suggest that Ras1V12 and RasV12S35 phenotypes are emulated by a hyperactivated form of Raf and suppressed by Raf, MEK, and MAPK mutants (Koh, 2002).

Studies in vertebrate cells and in Drosophila suggest that although Ras activation by receptor tyrosine kinases is blocked by the putative dominant-negative RasN17, Ras activation by PKC and the Ras1V12C40/PI3-K effect on cytoskeletal reorganization in fibroblasts are not. At the NMJ, Ras1N17 does not behave as a dominant negative. Thus, taken together, this analysis of NMJ structure in the different Ras strains suggests that Ras1 regulates the number of type I glutamatergic synapses in Drosophila and this regulation depends to a considerable extent on the activation of the MAPK pathway. Although activation of PI3-K and Ral-GDS-Ral by presumably PKC activation also points to a role for these pathways, their effect on NMJ growth is less prominent than the MAPK pathway (Koh, 2002).

Immunocytochemical studies of FasII immunoreactivity at synaptic terminals of MAPK gain- and loss-of-function mutants suggest that MAPK regulates levels of synaptic FasII, a cell-adhesion molecule that plays a key role in the maintenance and expansion of NMJs in Drosophila. This model was supported by experiments in which only type I synaptic FasII was immunoprecipitated. This was accomplished by using anti-DLG antibodies, because DLG binds directly to FasII at type I boutons but not at other bouton types. The immunoprecipitation experiments demonstrate that enhancing the levels of MAPK activity at synaptic terminals results in a reduction of type I synaptic FasII. Conversely, decreasing levels of MAPK activity results in an increase in type I synaptic FasII levels. These results are in agreement with the studies in Aplysia dissociated neurons, which show that ApMAPK is involved in the internalization of ApCAM (Koh, 2002).

Additional support for the idea that the changes in bouton number elicited by alterations in Ras1 and MAPK activity are mediated by alterations in FasII levels was demonstrated by examining the overall expression of FasII in MAPK gain- or loss-of-function alleles, examining the distribution of FasII within single synaptic boutons in relation to active MAPK, and using hypomorphic fasII mutants. The studies with rl mutants demonstrate that there is an inverse relationship between levels of synaptic FasII and MAPK activity. Furthermore, active MAPK localization coincides with regions of the bouton that have no or low FasII levels (Koh, 2002).

Two main functions of FasII in the regulation of synapse number have been demonstrated. (1) FasII is critically required for synapse maintenance: below threshold FasII levels, synaptic boutons are not maintained. (2) FasII operates by constraining synaptic growth, similar to the Aplysia system. Therefore, a decrease in FasII to a level still sufficient for maintenance results in an increase in synaptic arbor size. On the basis of this model, the following interpretation of the results is proposed. The dramatic decrease in FasII levels in the homozygous fasII mutant does not allow any influence of MAPK activity changes on NMJ structure. Similarly, when FasII levels are decreased to approximately one-half the wild-type levels (fasIIe76/+), an increase in MAPK activity does not induce an additional increase in bouton number, probably because an additional decrease in FasII compromises synaptic maintenance, thus preventing NMJ growth. However, the increase in FasII levels induced by a reduction of MAPK activity (rl10a/+) in a fasIIe76/+ background suppresses the increase in boutons observed in fasIIe76/+ alone. This result suggests that MAPK regulates FasII levels and exists upstream of FasII at signal transduction pathways that regulate the number of type I synaptic boutons (Koh, 2002).

Notably, the hypomorph rl10a/+ has no significant decrease in bouton number, although these mutants have a striking increase in FasII levels compared with wild-type controls. An explanation for this result is that FasII is a homophilic cell-adhesion molecule that is required both in the presynaptic and in the postsynaptic cell for function. If the Ras-MAPK pathway functions to regulate FasII at the presynaptic cell, as suggested by studies with cell-specific Gal4 drivers, then an asymmetric increase in FasII levels in the presynaptic cell alone may not have much of an effect. Previous studies also show that although the NMJ is very sensitive to a decrease in FasII levels, an increase in FasII over wild-type levels does not have much of an effect (Koh, 2002).

Although the results are consistent with a regulation of FasII-mediated synapse growth by the Ras-MAPK pathway, it is important to note that several other molecules in addition to FasII are involved in the regulation of synapse growth. Moreover, several studies suggest that many changes at the fly NMJ are compensated by yet unknown homeostatic mechanisms. Therefore, further understanding of these regulatory and compensatory signals will be necessary to fully explain these observations (Koh, 2002).

In conclusion, a signaling pathway intimately involved in the regulation of synaptic growth at the NMJ has been identified. Identification of the mechanisms involved in the activation of this pathway may provide valuable clues toward understanding the plasticity of this synapse (Koh, 2002).

Drosophila RSK negatively regulates bouton number at the neuromuscular junction

Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, the effect was investigated of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk-cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling (Fischer, 2009).

This study investigated the effect of rsk loss of function mutations in Drosophila, and found higher numbers of synaptic boutons in these mutants. The effect could be rescued by transgenic rsk expression. Vice versa overexpression of RSK reduced bouton numbers. Furthermore, removal of one allele of the Drosophila erk/rl gene normalized the effect of rsk loss of function on bouton formation, indicating that RSK mediates its effect through ERK/RL. Indeed, RSK and ERK/RL proteins interact directly with each other, and this interaction is abolished in the rlSem mutant. Furthermore, rlSem mutants show enhanced bouton numbers, similarly as rsk mutants, indicating that RSK negatively regulates ERK/RL activity at the NMJ and thus modulates bouton formation (Fischer, 2009).

A role of vertebrate RSK2 in inhibition of the RAS/ERK pathway has been proposed in several studies, but different underlying mechanisms have been suggested. In isolated mouse motoneurons, RSK2 is a negative regulator of axon growth by inhibiting ERK phosphorylation. In skeletal muscles of RSK2 knock-out mice, increased ERK activation has been observed. This could be explained by lack of inhibition of the ERK pathway via RAS guanine exchange factor SOS. In Drosophila, this inhibition seems not to occur through SOS. Knockdown of RSK2 leads to increased ERK phosphorylation in PC12 cells and cortical neurons. Moreover basal and 5HT2A receptor-mediated ERK 1/2 phosphorylation is increased in RSK2 knock-out fibroblasts These data are consistent with the current results showing that RSK interacts with ERK/RL and that this interaction leads to inhibition of ERK/RL activity in bouton formation at the NMJ (Fischer, 2009).

Previous studies on RSK and RL in the developing eye and wing imaginal disc provided evidence that RSK inhibits translocation of ERK/RL from the cytoplasm to the nucleus and thereby controls RL dependent gene transcription. However, the NMJ constitutes a separate part of the cell, and it is also conceivable that the effects of RSK and RL are mediated locally and do not involve nuclear translocation of these proteins. RSK seems to be present in the presynapse, but its distribution is diffuse and not restricted to active zones. This corresponds to the known distribution of RL in axon terminals. Thus, it is possible that RSK determines the localization of RL within synaptic boutons. Interestingly, an antibody that only recognizes active, phosphorylated RL showed a restricted localization to spots most likely corresponding to active zones. Thus one could speculate that RSK binds ERK/RL in axon terminals, thus inhibiting its activation, and only ERK/RL that is unbound can be activated by phosphorylation and move to active zones (Fischer, 2009).

In conclusion, these data indicate that RSK negatively regulates bouton formation at the NMJ, and that negative regulation of RL signaling is involved in this effect. Thus, Drosophila RSK seems to have a similar function as the RSK2 isoform in vertebrates. Therefore, the memory defects observed in flies, mice, and human CLS patients with mutations in rsk could be caused by dysregulated synapse architecture, as observed in the Drosophila model (Fischer, 2009).

Ribosomal s6 kinase cooperates with casein kinase 2 to modulate the Drosophila circadian molecular oscillator

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. This study shows that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, a physical interaction was demonstrated between S6KII and the casein kinase 2 regulatory subunit (CK2beta), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. It is proposed that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism (Akten, 2009).

The C-terminal kinase and ERK-binding domains of Drosophila S6KII (RSK) are required for phosphorylation of the protein and modulation of circadian behavior

A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. A previous study has reported that S6KII is required for normal circadian periodicity. This study reports a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. A role is demonstrated for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. Particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein (Tangredi, 2012).

This study utilized wild-type and mutant forms of S6KII in genetic rescue experiments to identify domains that are critical for the protein's function in circadian behavior. This is the first study to identify domains of S6KII (RSK) that are required, in vivo, for a behavioral function. Although in many cases S6KII isoforms were expressed at higher than normal levels in transgenic flies, it is not thought that results can be attributed to overexpression of the protein. Expression of wild-type S6KII at high levels has no discernible effects on circadian behavior or the phosphorylation pattern of S6KII. For example high level expression of a C-terminal kinase-dead mutant (S6KIIKm) does not rescue behavior nor phosphorylation defects observed at several sites including S357, a postulated PDK1 docking site within the N kinase domain. However there may be effects of S6KII overexpression that are not discernible in these molecular and behavioral assays (Tangredi, 2012).

In agreement with a previous study of fly development, this study shows that the S6KII N-terminal kinase is dispensable for its circadian function. This result contrasts with previous studies showing that RSK functions in the Ras/MAPK pathway as a kinase; it suggests that phosphorylation of downstream targets by the N-terminal kinase is not essential for modulation of the circadian clock. In support of a non-critical role for the N-terminal kinase, certain mutants that fail to rescue behavior nonetheless exhibit phosphorylation of S357, an event thought to activate RSK kinase activity. In addition, an S6KII mutant (S6KIIignΔ24−3) missing a large portion of the N-terminal region, including the N-terminal kinase, has been shown to partially rescue an S6KII-null mutant (Tangredi, 2012).

In contrast, the current studies emphasize the importance of S6KII C-terminal kinase activity for modulation of the Drosophila circadian clock. This is the first evidence, in either vertebrate or invertebrate systems, of a function for the S6KII C-terminal kinase that is independent of activation of the N-terminal kinase. It is also the first direct link between the C-terminal kinase and behavior. Heretofore, the only known function of the RSK C-terminal kinase was autophosphorylation, which leads to activation of the N-terminal kinase. The results suggest that either autophosphorylation serves an independent purpose (such as altering protein-protein interactions) or that the C-terminal kinase phosphorylates other proteins (Tangredi, 2012).

The C-terminal kinase was shown to promote phosphorylation of S515, a presumed autophosphorylation site and a residue within the hydrophobic motif site of AGC-type kinases. This region is important for stabilization of the catalytic domain of such kinases, including RSK, cAMP-dependent kinase and protein kinase C. It is noted that there is residual pS515 signal in a S6KII C-terminal kinase-dead mutant (S6KIIK597M) which may indicate that other kinases phosphorylate the site or that the mutant retains an undetectable amount of activity (Tangredi, 2012).

The work also suggests that S6KII S357 and T732 phosphorylation events are modulated by the C-terminal kinase. The N-terminal kinase is dispensable for circadian regulation, but nevertheless the data suggests that it is activated by the C-terminal kinase via phosphorylation of S357; in agreement with cell-based studies of RSK. The C-terminal kinase may promote S357 phosphorylation through recruitment of PDK1 or another factor. While the mechanism for C-terminal kinase modulation of T732 phosphorylation is unknown, it is possible that kinase activity that is stimulated by ERK binding feeds back to activate ERK phosphorylation of T732. This and other alterations may also involve the actions of phosphatases as there is undoubtedly a dynamic interplay between the two types of modifying enzymes. Of interest, pS515 levels are not altered in the T732A/T732E mutants, indicating that T732 phosphorylation is not a prerequisite for S515 phosphorylation (Tangredi, 2012).

There is a positive correlation between S6KII variants that rescue S6KIIign mutant behavior and robust phosphorylation of S515; this suggests that phosphorylation of this residue is essential for normal circadian behavior. It is noted, however, that while S515 phosphorylation is correlated with rhythmicity, it is not sufficient for normal circadian behavior. Therefore, pS515 may simply be indicative of a functional C-terminal kinase whose kinase activity is necessary for modulating circadian behavior via phosphorylation of other unknown targets. In addition, phosphorylation of this residue does not affect the phosphorylation of other S6KII domains; instead C-terminal kinase activity and modification of S515 may serve to alter S6KII protein conformation and relevant protein-protein interactions. While this study shows that C-terminal kinase activity is important for rhythmicity, the experiments do not exclude the idea that S6KII functions as a scaffold in the circadian system, analogous to its role in Drosophila eye and wing development (Tangredi, 2012).

ERK binding to S6KII is required for transgenic rescue of circadian behavior, as it is for rescue of Drosophila eye development phenotypes. Consistent with a role for ERK in this pathway, it was shown that phosphorylation of S6KII at T732 (a known ERK phosphorylation site on RSK) is required for rescue of behavioral rhythms. ERK phosphorylation of T732, previously demonstrated for RSK in mammalian cell-based assays, was verified in the fly by the observation that pT732 is reduced in ERK binding-deficient mutants. ERK binding may promote S6KII function by facilitating activation of the C-terminal kinase, as evidenced by the decreased autophosphorylation of ERK-binding mutants. Alternatively, ERK binding may alter S6KII localization and/or binding to clock-related proteins such as CK2, similar to S6KII's regulation of ERK in fly eye development. Whatever the precise mechanism, phosphorylation of T732 and S515 are likely to be important for ERK's interaction with S6KII and the regulation of circadian period (Tangredi, 2012).

It was demonstrated in fly photoreceptor cells that S6KII negatively regulates ERK by retaining it in the cytoplasm. Using immunostaining procedures, however, this study has shown that the localization pattern of ERK and diphosphorylated (activated) ERK are the same within PDF clock neurons (primarily cytoplasmic) in wild-type flies (w1118), S6KIIign-null mutants, and ERK-binding mutants (S6KIIign;timUG4>S6KIIR902A). Hence, ERK may bind to and activate S6KII in clock cells, but there is no evidence that S6KII regulates ERK localization in this cell type (Tangredi, 2012).

RSK protein is thought to be activated by a sequence of protein binding and phosphorylation events, based on cell-based investigations of the protein. More recent cell-based assays question the validity of this mode and give added relevance to the current studies as the first to examine this model in vivo (Tangredi, 2012).

This study provides the first evidence that phosphorylation/activation of Drosophila S6KII can occur in the absence of a strict sequence of binding and phosphorylation events, but it is noted that there is some dependence of certain events on others. Although there is extremely low pS515 immunoreactivity in C-terminal kinase and ERK-binding mutants, indicating that S515 phosphorylation is a downstream event, there is residual phospho-signal on this residue in such flies. Thus, ERK binding and C-terminal kinase activation may not be the only events contributing to S515 phosphorylation. Consistent with this idea, in vitro analysis of mammalian RSK has demonstrated that S380 phosphorylation (S515 in S6KII) and C-terminal kinase activation can occur in the absence of RSK-ERK interactions. Similarly, ERK binding and C-terminal kinase activity are not the only contributors to S6KII T732 phosphorylation because residual pT732 signal exists in mutants lacking these functions. The current results also indicate that neither ERK binding nor phosphorylation at S515 or T732 is essential for phosphorylation S357 although C-terminal kinase activity influences this event. This result is in agreement with mammalian cell-based studies demonstrating that N-terminal kinase activation is not fully dependent upon C-terminal kinase activity. Altogether, ERK-binding and C-terminal catalytic activity appear to play an important role in regulating phosphorylation of the S6KII protein, but the phosphorylation of individual sites is not absolutely required for the downstream phosphorylation of others (Tangredi, 2012).

Previous work indicated that S6KII modulates circadian function by negatively regulating the activity of the clock kinase CK2, via physical interaction with the CK2β subunit (Akten, 2009). Thus, it is possible that a prerequisite for the S6KII-CK2 interaction is activation of the S6KII C-terminal kinase or a conformational change in the protein resulting from ERK binding. CK2β may be a phosphorylation target of the S6KII C-terminal kinase (although there is no evidence of this), and this would provide a mechanism by which S6KII could regulate CK2 activity. Alternatively, a change in S6KII conformation might regulate interaction with CK2, thus modulating the previously documented effects of the kinases on the PER-based clock. Finally, the possibility exists that S6KII regulates circadian clock function through a CK2-independent pathway. Further analysis of the S6KII binding partners and substrates may yield insights about the precise role of the C-terminal kinase and ERK-binding domains in circadian regulation (Tangredi, 2012).

Impact of ERK activation on fly survival and Wolbachia-mediated protection during virus infection

Elevated levels of reactive oxygen species (ROS) provide protection against virus-induced mortality in Drosophila. In addition to contributing to oxidative stress, ROS are known to activate a number of signaling pathways including the extracellular signal-regulated kinases (ERK) signaling cascade. It was recently shown that ERK signaling is important for resistance against viral replication and invasion in cultured Drosophila cells and the gut epithelium of adult flies.bUsing a Drosophila loss-of-function ERK (rolled) mutant this study demonstrated that ERK is important for fly survival during virus infection. ERK mutant flies subjected to Drosophila C virus (DCV) oral and systemic infection were more susceptible to virus-induced mortality as compared to wild type flies. It was demonstrated experimentally that ERK activation is important for fly survival during oral and systemic virus infection. Given that elevated ROS correlates with Wolbachia-mediated antiviral protection, the involvement of ERK in antiviral protection was also investigated in flies infected by Wolbachia. The results indicate that ERK activation is increased in the presence of Wolbachia but this does not appear to influence Wolbachia-mediated antiviral protection, at least during systemic infection (Wong, 2016).

Park, S.M., Park, H.R. and Lee, J.H. (2017). MAPK3 at the autism-linked human 16p11.2 locus influences precise synaptic target selection at Drosophila larval neuromuscular junctions. Mol Cells [Epub ahead of print]. PubMed ID: 28196412

MAPK3 at the autism-linked human 16p11.2 locus influences precise synaptic target selection at Drosophila larval neuromuscular junctions
Proper synaptic function in neural circuits requires precise pairings between correct pre- and post-synaptic partners. Errors in this process may underlie development of neuropsychiatric disorders, such as autism spectrum disorder (ASD). Development of ASD can be influenced by genetic factors, including copy number variations (CNVs). This study focused on a CNV occurring at the 16p11.2 locus in the human genome and investigated potential defects in synaptic connectivity caused by reduced activities of genes located in this region at Drosophila larval neuromuscular junctions, a well-established model synapse with stereotypic synaptic structures. A mutation of rolled, the Drosophila homolog of human mitogen-activated protein kinase 3 (MAPK3) at the 16p11.2 locus, causes ectopic innervation of axonal branches and their abnormal defasciculation. The specificity of these phenotypes was confirmed by expression of wild-type rolled in the mutant background. Albeit to a lesser extent, ectopic innervation patterns were also observed in mutants defective in Cdk2, Gaq, and Gp93, all of which are expected to interact with Rolled MAPK3. Further genetic analysis in double heterozygous combinations reveals a synergistic interaction between rolled and Gp93. In addition, results from RT-qPCR analyses indicate consistently reduced rolled mRNA levels in Cdk2, Gaq, and Gp93 mutants. Taken together, these data suggest a central role of MAPK3 in regulating the precise targeting of presynaptic axons to proper postsynaptic targets, a critical step that may be altered significantly in ASD (Park, 2017).

rolled/MAPK: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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