rolled/MAPK
The Torso signal transduction pathway exhibits two opposite effects on the activity of the Bicoid (Bcd) morphogen: (1) Bcd function is repressed by Torso (Tor) at the anterior pole of the embryo leading to a retraction of the expression of many Bcd targets from the most anterior region of the embryo, where the Tor tyrosine kinase receptor is activated, and (2) Bcd function is strengthened by Tor in a broader anterior region, as indicated by a shift of the posterior border of Bcd targets toward the anterior pole in embryos deprived of Tor activity. Anterior repression of Bcd targets is not observed in embryos lacking maternal contribution of D-sor, which acts downstream of Tor and encodes a MAP-kinase kinase. This indicates that the Ras signaling cascade is directly involved in this process, although the known transcriptional effectors of the Tor pathway, tll and hkb, are not. Bcd is a good in vitro substrate for phosphorylation by MAP-kinase (MK); phosphorylation of the protein occurs in vivo on MK sites. Examination of Bcd sequence reveals three optimal (S165, T200 and T353) and seven weak (T188, T193, S195, T197, S343, S359 and S439) consensus sites for phosphorylation by MK. Six of these sites are concentrated in the serine/threonine (S/T)-rich domain that follows the HD. This domain also contains a PEST sequence that has been implicated in the degradation of proteins with short half-lives. The four remaining MK sites are located within the activation domain in the C-terminal part of the protein. In the presence of a Bcd mutant that can no longer be phosphorylated by MK, expression of Bcd targets remains repressed by Tor at the pole while the strengthening of Bcd activity is reduced. These experiments indicate that phosphorylation of Bcd by MK is likely to be required for the Tor pathway to induce its full positive effect on Bcd. This suggests that Tor signaling acts at a distance from the anterior pole by direct modification of the diffusing Bcd morphogen (Janody, 2000).
Tailless acts as a repressor of Kruppel and knirps in the central domain of the recently fertilized embryo. Groucho acts throughout the embryo to repress the repressor of Kruppel and knirps, allowing the expression of these gap genes in the central domain of the embryo. Patterning of the non-segmental termini of the Drosophila embryo depends on signaling via the Torso receptor tyrosine kinase (RTK). Activation of Torso at the poles of the embryo triggers expression of the terminal zygotic gap genes tailless (tll) and huckebein (hkb). The Groucho (Gro) corepressor acts in this process to confine terminal gap gene expression to the embryonic termini. Embryos lacking maternal gro activity display ectopic tll and hkb transcription; in turn, tll then leads to lack of abdominal expression of the Kruppel and knirps gap genes. torso signaling permits terminal gap gene expression by antagonizing Gro-mediated repression. Groucho-mediated repression of tailless is relieved by the torso pathway suggesting that Groucho is the nuclear target for MAP kinase signaling. It is suggested that Groucho functions as a corepressor along with an unknown protein unrelated to Hairy, since Groucho mediated repression takes place in the absence of known Hairy-related bHLH proteins. Thus, the corepressor Gro is employed in diverse developmental contexts and, probably, by a variety of DNA-binding repressors (Paroush, 1997).
Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).
To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rho were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis. The effect of Egfr on neuroblast formation was monitored by the neuroblast marker Snail. Anti-Sna staining reveals three columns of SI neuroblasts in the control embryo: the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells. In Egfr and rho mutants, Sna-positive neuroblasts in the intermediate position are frequently missing, with a higher frequency of loss in Egfr embryos. In rho mutant embryos, the frequency of the loss of intermediate column neuroblasts is variable among embryos (Yagi, 1998).
To further examine the effect of the loss of Egfr signaling on the late events of neurogenesis, the progeny was traced for one of the intermediate neuroblasts, NB4-2. NB4-2 gives rise to the RP2 motor neurons, which can be identified by the expression of Even-skipped (Eve) and its unique position. Loss of RP2 neurons in stage 13 is observed (over half the cases examined) with the frequency of loss slightly higher in Egfr than in rho mutants, reflecting the earlier defect in neuroblast formation in stage 9. It is known that the Ras-MAPK signaling cascade is the major target of Egfr in many tissues. To understand whether Ras-MAPK signaling also mediates the Egfr signal in the neuroectoderm and to determine the relative contribution of each component of the pathway, the expression of esg and sc was examined in embryos lacking one of the Ras-MAPK signaling components. The phenotype of mutants lacking either Sos, Ras1, Draf or Dsor1 was examined. As in wild-type embryos, embryos mutant for any of the four genes examined express esg in three separate domains: procephalic neurogenic region, amnioserosa and neuroectoderm. In all cases, the anterior limit of the procephalic expression and the posterior limit of neuroectodermal expression are expanded to the terminus, consistent with the fact that Ras-MAPK is required for the terminal fate specification controlled by Torso receptor tyrosine kinase. All mutants exhibit specific defects within the neuroectoderm where esg expression is derepressed in the intermediate column. Essentially the same phenotype is also observed with sc expression, suggesting the loss of Ras-MAPK signaling has the same consequence as the loss of Egfr. All four Ras pathway mutants show, qualitatively, the same phenotype in the neuroectoderm. The neuroectoderm phenotype in Ras1 mutants is not rescued by a paternal copy of the wild-type gene, suggesting that a relatively high dose of the Ras signal is required for repression of esg and sc in the neuroectoderm (Yagi, 1998).
Rhomboid (rho) is initially expressed in the medial half of neuroectoderm, but repression of esg, ac and sc transcription by Egfr and Ras-MAPK occurs only in the intermediate column, posing a question as to whether or not the site of MAPK activation and the site of transcriptional repression exactly correspond. The spatial and temporal pattern of MAPK activation has been described by the use of an antibody that specifically reacts with the phosphorylated and activated form of MAPK (diphospho-MAPK=dpMAPK), which shows that dpMAPK is distributed in a broad domain in the neuroectoderm in stage 5-7 embryos. dpMAPK is distributed in an 8- to 10- cell-wide area in the neuroectoderm in stage-5 embryos and becomes restricted to the ventral region at the end of gastrulation. This rapidly evolving pattern of dpMAPK expression made it difficult to determine the exact correlation between distribution of dpMAPK and the DV subdomains in the neuroectoderm. A protocol was used to double label embryos with dpMAPK and antisense RNA probes to study the spatiotemporal relationship between expression of dpMAPK, its activator Rhomboid (Rho) and its downstream target, esg. Initial expression of dpMAPK overlaps with that of Rho in stage-5 embryos; dpMAPK expression remains in this broad domain when Rho expression became restricted to the medial column at gastrulation in stage 6, and finally narrows down to a 2- to 3-cell-wide stripe abutting the stripe of Rho at stage 7. Comparison with the mesodermal marker sna shows that the ventral border of dpMAPK expression abuts the neuroectoderm-mesoderm border. Examination of histochemically stained material reveals a sharp ventral border of dpMAPK expression, which gradually declines in the dorsal direction, resembling the pattern of Rho expression. In Egfr mutant embryos, dpMAPK staining is not detectable. These results demonstrate that MAPK activation in the neuroectoderm is dependent on Egfr and follows the spatial expression pattern of Rho, but persists for some time after termination of Rho transcription. The latter observation may reflect perdurance of Rho or its target protein, Spitz (Spi). Alternatively, a ligand other than Spi, such as Vein, might be activating Egfr. The dorsal limit of dpMAPK expression was determined relative to the three separate columns of neuroectoderm revealed by esg expression. In stage 5, the dorsal limit of dpMAPK reaches halfway within the intermediate column and subsequently retracts to the medial column in stage 6 and 7. These data indicate MAPK is activated at least in the ventral half of the intermediate column of the neuroectoderm when it is required to repress transcription of esg. It is concluded that transcription of esg is repressed by a marginal level of MAPK activation (Yagi, 1998).
Why does the high level of dpMAPK in the medial column fail to repress transcription of esg, ac and sc? One possibility is that a factor is present in the medial column that antagonizes or overcomes the events downstream of dpMAPK. A candidate for such a gene is vnd, which is expressed in the medial column in late stage 5 and is required for expression of ac. Expression of esg and sc was examined in vnd null mutant embryos: their expression in the medial column was found to be lost. To understand how vnd controls gene expression in the medial column, a target for vnd was sought in the cis-regulatory regions of an esg enhancer. Expression of esg is regulated by the neurogenic enhancer, which can be divided into two regions, the activator region, which mediates activation in the entire neuroectoderm, and the repressor region, which mediates Egfr-dependent repression. Expression of the esg-lacZ fusion genes was examined in the vnd mutant background. The construct esg-lacZ D1 containing the complete neurogenic enhancer reproduces neuroectodermal expression of esg and is regulated by vnd in the same manner as esg. In contrast, the construct esg-lacZ D5 lacks the repressor region for the Egfr-mediated regulation and is expressed in all three columns. Evidence is provided that vnd does not regulate esg-lacZ D5 and that the target site for vnd regulation is included in the repressor region. vnd is also shown not to be involved in activation of esg or Egfr; rather, it works to counteract the negative effect of Egfr (Yagi, 1998).
Given the results of the present work showing that vnd counteracts the negative regulatory effect of Egfr, a model is proposed for the DV structuring of the neuroectoderm. A gradient of nuclear localized Dorsal protein induces expression of dorsoventrally regulated genes such as dpp, sna, and twi, which determine the extent of the neuroectoderm, and the nested expression domains of rhomboid and vnd. rho determines the domain of MAPK activation, which covers the medial and intermediate columns. vnd is expressed in the medial column where it counteracts the Egfr signal to allow expression of esg. Thus the three columns in the stage 5-6 neuroectoderm are distinguished by unique combinations of activated MAPK and vnd expression. In the lateral column, neither of them are activated or expressed, and esg transcription is activated by default. In the intermediate column, MAPK is activated and represses esg transcription. In the medial column, vnd counteracts activated MAPK to allow the default pathway to activate esg transcription. It is possible that proneural genes are also regulated by the same mechanism. Loss of the Egfr signal leaves two domains, one with and the other without expression of vnd, the pattern likely to be reflected in the appearance of only two neuroblast columns in the later stage. Thus it is proposed that the primary role of Egfr signal in this stage is to define the intermediate domain to the neuroectoderm which is otherwise separated into two domains. It is possible that Egfr signal and vnd have later roles in promoting neuroblast formation in the intermediate and medial columns, respectively (Yagi, 1998).
The expression of Jun in the eye imaginal disc correlates temporally and spatially with the determination of neuronal photoreceptor fate. Expression of dominant negative forms of Jun in photoreceptor precursor cells results in dose-dependent loss of photoreceptors in the adult fly. Conversely, localized overexpression of Jun in the eye imaginal disc can induce the differentiation of additional photoreceptor cells. Furthermore, the transformation of nonneuronal cone cells into R7 neurons elicited by constitutively active forms of sevenless, Ras1, Raf, and MAP kinase is relieved in the presence of Jun mutants. These results demonstrate a requirement of Jun downstream of the sevenless/ras signaling pathway for neuronal development in the Drosophila eye (Bohmann, 1994).
Phosphorylation of JUN by the MAP kinase Rolled regulates photoreceptor differentiation. In fact, DJUN can sequester Rolled protein from a crude extract, indicating a specific interaction. D-JUN is phosphorylated on three conserved MAPK sites. A Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which phosphorylation sites are replaced by phosphate-mimetic Asp residues can promote photoreceptor differentiation (Peverali, 1996).
The strong eye ablation phenotype in Drosophila, caused by expressing head involution defective under the control of an eye-specific promoter, was used to perform a genetic screen aimed at identifying components that regulate and mediate Hid activity. Mutations in genes that regulate the EGF receptor (EGFR)/Ras1 (Ras oncogene at 85D) pathway were recovered as strong suppressors of Hid-induced apoptosis. The survival effect of the EGFR/Ras1 pathway is specific for Hid-induced apoptosis, since neither Reaper- nor Grim-induced apoptosis is affected by the EGFR/Ras1 pathway. The Ras1 pathway has been shown to inhibit Hid activity apparently by the direct phosphorylation of Hid by MAPK (Rolled). Alteration of the MAPK phosphorylation sites within the HID sequence blocks the survival signals generated by constitutively activate Ras1 and constitutively active MAPK. It is concluded that the hid gene in Drosophila provides a mechanistic link between the survival activity of Ras1 and the apoptotic machinery. Post-translational modification of Hid is a survival signal regulating Hid activity (Bergmann, 1998).
The Dorsal nuclear gradient initiates the differentiation of the mesoderm, neuroectoderm, and dorsal ectoderm by activating and repressing gene expression in the early Drosophila embryo. This gradient is organized by a Toll signaling pathway that shares many common features with the mammalian IL-1 cytokine pathway. A second signaling pathway, controlled by the Torso receptor tyrosine kinase, also modulates DL activity. The Torso pathway selectively masks the ability of DL to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of Torso, such as D-raf and the Rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and Torso pathways are both active only at the embryonic poles, and consequently, target genes (zerknüllt and decapentaplegic), repressed in middle body regions, are expressed at these sites. Constitutive activation of the Torso pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor (Rusch, 1994).
Activation of the Torso RTK at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. The tor response element (tor-RE) in the tll promoter indicates that the key activity modulated by the tor RTK pathway is a repressor present throughout the embryo. The tor-RE has been mapped to an 11-bp sequence. The proteins GAGA and NTF-1 (also known as Elf-1, product of the grainyhead gene) bind to the tor-RE. NTF-1 can be phosphorylated by Rolled/MAPK. tll expression is expanded in embryos lacking maternal NTF-1 activity. These results make NTF-1 a likely target for modulation by the tor RTK pathway in vivo. Thus activation of the tor RTK at the poles of the embryos leads to inactivation of the repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo (Liaw, 1995).
RNA polymerase (RNAP) II is a multisubunit enzyme composed of several different subunits. Phosphorylation of the C-terminal domain (CTD) of the largest subunit is tightly regulated. In quiescent or in exponentially growing cells, both the unphosphorylated (IIa) and the multiphosphorylated (IIo) subunits of RNAP II are found in equivalent amounts as the result of the equilibrated antagonist action of protein kinases and phosphatases. In both Drosophila and mammalian cells, heat shock markedly modifies the phosphorylation of the RNAP II CTD. Mild heat shocks result in dephosphorylation of the RNAP II CTD. This dephosphorylation is blocked in the presence of actinomycin D, and the CTD dephosphorylation observed in the presence of protein kinase inhibitors. Thus, heat shock might inactivate CTD kinases which are operative at normal growth temperatures, as some protein kinase inhibitors do. In contrast, severe heat shocks are found to increase the amount of phosphorylated subunit independently of the transcriptional activity of the cells. Mild and severe heat shocks activate protein kinases, which then phosphorylate the CTD fused to beta-galactosidase, both in vitro and in vivo. The heat-shock-activated CTD kinases are identified as p42mapk and p44mapk. The weak CTD kinase activation occurring upon mild heat shock might be insufficient to compensate for the heat inactivation of the already existing CTD kinases. However, under severe stress, the MAP kinases are strongly heat activated and might prevail over the phosphatases. A survey of different cells and different heat-shock conditions shows that the RNAP II CTD hyperphosphorylation rates follow the extent of MAP kinase activation. These observations lead to the proposal that the RNAP II CTD might be an in vivo target for the activated p42mapk and p44mapk MAP kinases (Venetianer, 1995).
The fate of the R7 photoreceptor cell in the developing eye of Drosophila is controlled by the Sevenless (Sev) receptor tyrosine kinase. Sev activates a highly conserved signal transduction cascade involving the proteins Ras1 and Raf and the Rolled/mitogen-activated protein kinase. The ETS domain protein encoded by the P2 transcript of the pointed gene is a nuclear target of this signaling cascade that acts downstream of Rolled. The PntP2 protein is phosphorylated by Rolled in vitro at a single site and this site is required for its function in vivo. Rolled controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family: Anterior open/Yan and PntP2 (Brunner, 1994b).
R7 photoreceptor fate in the Drosophila eye is induced by the activation of the Sevenless receptor tyrosine kinase and the RAS/MAP kinase signal transduction pathway. Expression of a constitutively activated JUN isoform in ommatidial precursor cells is sufficient to induce R7 fate independent of upstream signals normally required for photoreceptor determination. JUN interacts with the ETS domain protein Pointed to promote R7 formation. This interaction is cooperative when both proteins are targeted to the same promoter and is antagonized by another ETS domain protein, YAN, a negative regulator of R7 development. Furthermore, phyllopod, a putative transcriptional target of RAS pathway activation during R7 induction, behaves as a suppressor of activated JUN. Taken together, these data suggest that JUN and Pointed act on common target genes to promote neuronal differentiation in the Drosophila eye, and that phyllopod might be such a common target (Treier, 1995).
Anterior open/Yan has been postulated to act as an antagonist of the proneural signal mediated by the sevenless/Ras1/MAPK pathway. The eight MAPK phosphorylation consensus sites of Yan were mutagenized and the effects of overexpressing the mutant protein in transgenic flies and transfected S2 cultured cells were examined. Phosphorylation by MAPK affects the stability and subcellular localization of Yan, resulting in rapid down-regulation of Yan activity. Furthermore, MAPK-mediated down-regulation of Yan function appears to be critical for the proper differentiation of both neuronal and nonneuronal tissues throughout development, suggesting that Yan is an essential component of a general timing mechanism controlling the competence of a cell to respond to inductive signals (Rebay, 1995).
The evolutionarily conserved Ras/mitogen-activated protein kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors. A new component of this signal transduction pathway called Mae (for modulator of the activity of Ets) has bee cloned and characterized. Mae is a signalling intermediate that directly links the MAPK signalling pathway to its downstream transcription factor targets. Phosphorylation by MAPK of the critical serine residue (Ser 127) of the Drosophila transcription factor Yan depends on Mae, and is mediated by the binding of Yan to Mae through their Pointed domains. This phosphorylation is both necessary and sufficient to abrogate transcriptional repression by Yan. Mae also regulates the activity of the transcriptional activator Pointed-P2 by a similar mechanism. Mae is essential for the normal development and viability of Drosophila, and is required in vivo for normal signalling of the epidermal growth factor receptor. This study indicates that MAPK signalling specificity may depend on proteins that couple specific substrates to the kinase (Baker, 2001).
Phosphorylation of transcription factors by MAPK is a key link between cell signalling and the control of gene expression. The Ets family of transcription factors regulates cell growth and differentiation, and the activities of many of its 35 members are modulated through phosphorylation by MAPK4. To identify signalling intermediates, proteins were isolated that interact with the Drosophila Ets transcription factor Yan. Yan functions downstream of several receptor tyrosine kinase (RTK) pathways, where it regulates the differentiation of many precursor cells. Experiments in tissue-culture cells suggest that Yan functions as a transcriptional repressor whose activity is attenuated rapidly by MAPK phosphorylation (Baker, 2001).
Using full-length yan complementary DNA as a bait, a yeast two-hybrid screen was conducted of an 18-h Drosophila embryo library, from which a full-length cDNA clone was recovered. The only region of homology between Yan and the predicted Mae protein is a Pointed (Pnt) domain located at the carboxy terminus. The Pnt domain (also called the SAM domain) defines a subfamily of Ets proteins, including Ets-1, Ets-2, GABP and TEL from vertebrates, and Drosophila Yan and Pnt-P2. It is also found in Polycomb-group proteins, where it mediates protein-protein interactions, and in MAPK kinase kinases that are components of the MAPK cascade (Baker, 2001).
Since Yan represses transcription by binding to consensus Ets DNA-binding sites, whether Mae interferes with Yan binding to DNA in a gel mobility shift assay was tested. Mae does not bind to the consensus Ets DNA-binding site, but it prevents Yan from doing so. This inhibition is mediated through the Pnt domain-dependent binding of Mae to Yan. DNA binding by Yan requires a functional Ets DNA-binding domain, but not intact phosphorylation sites or a functional Pnt domain, because YanACT (in which all nine MAPK consensus sites are mutated to alanine) binds the DNA site normally, as does Yan(G84P) in which an amino acid conserved in all Pnt domains is mutated (Baker, 2001).
An assay was established of transcriptional repression by Yan. Because Drosophila S2 cells express mae endogenously, the yan and mae constructs were expressed in Cos-7 cells. Yan represses the activity of a TK-luciferase reporter tenfold. Repression depends on the Ets DNA consensus site, and on the Ets DNA-binding domain in Yan. As expected, Mae alone does not affect reporter activity because it lacks an Ets DNA-binding domain (Baker, 2001).
RTK signalling downregulates Yan activity through phosphorylation mediated by Ras and the Erk/Rolled MAPK. Expression of a constitutively active form of Ras (12H-Ras) completely abrogates transcriptional repression by Yan, but only in the presence of Mae. Consequently, Mae is required for Ras to inactivate Yan in Cos-7 cells. An excess of Mae alone can reduce the repressive effect of Yan (3-fold), presumably through formation of Mae-Yan heterodimers. However, low quantities of Mae, which alone inhibit the repressive effect of Yan by 30%, are sufficient to mediate the complete, Ras-dependent inactivation of Yan. Furthermore, the non-phosphorylatable YanACT mutation is insensitive to Ras/Mae-induced inhibition, indicating that Mae inhibits Yan by promoting its phosphorylation by MAPK. Indeed, Yan mutants that cannot bind Mae -- Yan(G84P) and Yan(46-107) -- are also resistant to inactivation by Ras/Mae (Baker, 2001).
Although Yan has nine sites of MAPK phosphorylation, Ser 127 is the critical regulatory site of Yan because mutation to alanine creates a constitutive repressor that is refractory to downregulation by Ras signalling6. To test whether Mae mediates phosphorylation of Ser 127 of Yan, in vitro phosphorylation of Yan by activated Erk was examined. Activated Erk phosphorylates wild-type Yan independently of Mae, and, as expected, is unable to phosphorylate the constitutive repressor YanACT, which has all nine MAPK phosphorylation sites mutated. Notably, Yan(S127), in which all consensus phosphorylation sites except Ser 127 are mutated to alanine, is resistant to phosphorylation by Erk alone, even with very high amounts (up to 1 microg) of the kinase. Yan(S127) is efficiently phosphorylated by Erk and Mae together, however, indicating that Mae is needed for phosphorylation of this Ser 127 residue (Baker, 2001).
This phosphorylation depends on the Pnt domain of Yan, indicating that the ability of Mae to promote Erk-directed phosphorylation of Ser 127 is dependent on binding of Mae to Yan. Yan(S127) represses transcription of the luciferase reporter as efficiently as the wild-type protein, and is also completely susceptible to inactivation by 12H-Ras/Mae. Therefore, Mae is required for MAPK to abolish transcriptional repression by Yan, by mediating phosphorylation of the critical regulatory residue Ser 127 (Baker, 2001).
Inhibition of Yan repression by RTK signalling in Drosophila is accompanied by activation of a transcription factor encoded by the pointed (pnt) gene. pnt encodes two alternative Ets transcription factors: P1, a constitutively active transcriptional activator whose expression is induced by Ras/MAPK signalling; and P2, a transcriptional activator that, like Yan, contains an amino-terminal Pnt domain and whose activity is stimulated through phosphorylation by the Ras/MAPK pathway. Activation of Pnt-P2 by Erk also seems to depend on Mae: the two proteins physically associate and this interaction is dependent on the Pnt domain. Pnt-P2 is a weak activator in this transcription assay and is not stimulated by Ras alone. However, Ras and Mae together augment its transcriptional activity fourfold. Thus, Mae also regulates Pnt-P2 activity, presumably by promoting its phosphorylation by MAPK (Baker, 2001).
These results show that Mae is needed in cultured cells to permit regulation of Ets transcription factor activity by Erk. This requirement was not evident in previous experiments on Yan because Drosophila S2 cells were used in which endogenous mae is expressed at high levels (Baker, 2001).
To study Mae function in vivo, in situ hybridization was used to examine Maeexpression in early Drosophila embryos. In stage 6-7 embryos, mae is expressed in bilaterally symmetric anteroposterior stripes, 3-4 cells wide, that flank the ventral midline. These stripes of expression narrow to a width of two cells and one cell adjacent to the midline by stage 9 and stage 11, respectively. mae expression is very similar to the expression pattern of epidermal growth factor receptor (EGFR) signalling regulators such as rhomboid (rho), vein, argos, yan and pnt, and it marks the ventral neurectodermal zone that is patterned by EGFR signalling. mae is subsequently expressed in tracheal pits, in ventral denticle domains, and in areas such as the optic lobe and the medial domains of the brain, all of which are sites of EGFR activity (Baker, 2001).
To investigate further the in vivo role of mae, the enhancer-trap lines l(2)k06602 and l(2)k12907, which contain single P-element insertions that are responsible for the lethal phenotype, were analyzed. These P-elements insert into the 5' untranslated region (UTR) of the mae gene [0.8-kilobases (kb) and 50-base-pairs (bp) upstream of the mae initiation codons in l(2)k0660 and l(2)k12907, respectively]. ß-Galactosidase in early l(2)k06602 and l(2)k12907 embryos is, like mae, expressed in the ventral neurectoderm, tracheal pits and ventral denticle belts. Furthermore, homozygous mae mutant embryos (25% of embryos derived from either heterozygous l(2)k06602 or l(2)k12907 parents) lack detectable mae expression. These results show that the P-element insertions affect development because they disrupt mae expression (Baker, 2001).
A role for Mae in EGFR signalling is further supported by certain features of the mae mutant phenotype. Embryonic patterning is affected in mae mutant embryos, especially towards the midline of the anterior abdominal segments. Ventral denticle belts are thinner, and rows of denticles, particularly the first, are missing or misorientated. maek12907 and maek06602 embryos each show a similar phenotype when hemizygous for Df(2R)PC4, a deficiency for the region, indicating that the patterning defects result from lack of mae expression. Preferential disruption of midline patterning in anterior abdominal segments is reminiscent of the phenotype of rho and pnt mutant larvae (Baker, 2001).
mae seems to be a downstream component of the EGFR signalling pathway. Its pattern of expression depends on Rho, the spatially restricted limiting component of EGFR signalling, but is not required for rho expression. Furthermore, in the l(2)k12907 enhancer-trap line in which mae expression is abolished, lacZ marks the mae transcriptional domains (and thereby domains of EGFR signalling), and this domain is broadened along the ventral midline. Similarly, the expression domain of argos is broadened in early stage mae mutant embryos, although levels of expression, notably in later stage embryos, seem to be reduced. argos is a downstream target of EGFR signalling whose expression is also disrupted in Drosophila embryos that are mutant for regulators of EGFR signalling such as Ras, Yan, Pnt and Rho (Baker, 2001).
The above experiments show that Mae mediates inactivation of Yan by MAPK phosphorylation of the critical regulatory Ser 127 residue. Presumably, binding of Mae causes a local change in the conformation of Yan, which exposes Ser 127 to MAPK. Further evidence for this idea is that Mae binding to the Pnt domain in Yan also affects the ability of Yan to bind DNA through its Ets domain. Erk can associate with Mae and Yan in GST pull-down assays but, unlike Mae binding to Yan, this interaction is weak and is not evident in co-precipitation assays. It is proposed that Mae binding to Yan or Pnt-P2 allows MAPK to phosphorylate their critical residues in a transitory ternary enzyme-substrate complex (Baker, 2001).
This is the first report of a requirement for an intermediary protein linking MAPK to its substrate, and it suggests a mechanism for achieving tissue-specific responses to the generic RTK/Ras/MAPK signalling pathways; that is, determination of the MAPK substrate through tissue-specific adapter/coupling proteins. Cells will respond differently to MAPK according to the adapter/coupling proteins that they express. Future work will help test whether phosphorylation of other MAPK substrates also depends on adapter/coupling proteins (Baker, 2001).
A key question in developmental biology is how growth factor signals are integrated to generate pattern. This study investigated the integration of the Drosophila BMP and Wingless/GSK3 signaling pathways via phosphorylations of the transcription factor Mad. Wingless was found to regulate the phosphorylation of Mad by GSK3 in vivo. In epistatic experiments, the effects of Wingless on wing disc molecular markers (senseless, distalless and vestigial) were suppressed by depletion of Mad with RNAi. Wingless overexpression phenotypes, such as formation of ectopic wing margins, were induced by Mad GSK3 phosphorylation-resistant mutant protein. Unexpectedly, Mad phosphorylation by GSK3 and MAPK was found to occur in segmental patterns. Mad depletion or overexpression produced Wingless-like embryonic segmentation phenotypes. In Xenopus embryos, segmental border formation was disrupted by Smad8 depletion. The results show that Mad is required for Wingless signaling and for the integration of gradients of positional information (Eivers, 2009; full text of article).
Transgenic flies expressing forms of Mad resistant to GSK3 phosphorylation displayed high BMP and Wg signaling phenotypes. Mad contains 74 serines/threonines, yet phosphorylation-resistant mutations of a single MAPK or of two GSK3 sites generated hyperactive transcription factors. Previous work in Drosophila had identified that phosphorylation by the Nemo/NLK kinase in the MH1 domain of Mad inhibits its activity, and that a neomorphic human Smad4 mutation can produce Wg-like phenotypes when overexpressed in the wing. During Drosophila early embryogenesis, Mad linker phosphorylations tracked the priming activity of MAPK/EGFR, particularly in the ventral side, suggesting that Mad may be regulated independently of dorsal Dpp signals. Drosophila EGFR activates MAPK in a broad ventral region which corresponds to the neurogenic ectoderm. Although this study focused on the role of Wg/GSK3 on Mad regulation, the priming phosphorylation for GSK3 is provided by EGFR signaling and is critical for Mad to be polyubquitinated and degraded in the centrosome (Eivers, 2009).
Mad MGM (Mad GSK mutant; see Phosphorylation-Resistant Mad Proteins are Hyperactive), which mimics Mad receiving a maximal Wg signal, phenocopied known Wg overexpression phenotypes. In the wing, MGM caused the formation of ectopic rows of Senseless-expressing cells, sensory bristles, and entire ectopic wing margins. In larval cuticles, MGM caused the reduction of ventral denticle belts, which were replaced by naked cuticle regions, an indicator of Wg signaling. The mad gene product was demonstrated to be involved in Wg signaling in multiple in vivo assays. In the wing disc, Wg overexpression strongly increased senseless, distalless, optomotor blind, and vestigial transcripts, and co-expression of Mad RNAi inhibited this effect, without affecting Wg expression levels. The induction of ectopic neurogenic ectoderm tissue positive for SoxNeuro by RNAi was epistatic to the inhibition of SoxNeuro expression caused by Wg overexpression. Taken together, these results suggest that Mad is a required component for several Wg signaling events in Drosophila (Eivers, 2009).
Segmentation phenotypes were observed when Mad RNAi was expressed maternally using the pUASp vector. Segment fusions were generated in which larval naked cuticle was replaced by large denticles of the same type (row 5) as those seen in Wg nulls. In gain-of-function experiments, overexpression of GSK3-resistant Mad caused denticle belts to be replaced by naked cuticle, mimicking Wg signaling. Thus, depletion or overexpression of Mad generated Wg-like phenotypes, indicating that Mad functions in the Wg signaling pathway during segmental patterning (Eivers, 2009).
The MAPK pathway, which during Drosophila embryonic segmentation is regulated by EGFR activity, would decrease the duration of the Mad signal by promoting Mad polyubiquitination and degradation. The EGFR-activating genes rhomboid and spitz are activated in the anterior of each segment and Wg in the posterior border of the anterior compartment. Wg/Wnt signals would increase the duration of the signal by inhibiting GSK3 phosphorylations, generating a double gradient of GSK3 and MAPK activities that would regulate Mad stability and signaling within each segment. This may occur in a Dpp-independent fashion, but it is also possible that BMP signals might be active during larval segmentation, since the expression of the BMP receptor thickveins has a segmental pattern of expression. In addition, Dpp is expressed in the ectoderm during segmentation stages, and its promoter contains segmentation modulation elements (Eivers, 2009).
Finding a role for Mad in segmentation was remarkable, because this process has been extensively studied in Drosophila genetic screens and Mad had not been previously implicated as part of the segmentation machinery. This new role for Mad can be explained by the fact that Mad appears to also function independently of Dpp and that the Mad10 and Mad12 null alleles are nulls only for the BMP pathway. This persistence of a Mad linker regulation by phosphorylation could explain results in the literature showing that Mad10 mutant clones can result in Wg-like effects in the Drosophila wing. Overexpression of Mad12 synthetic mRNAs mutated in the GSK3 phosphorylation sites have strong posteriorizing activity in Xenopus embryos. This indicates that Mad mutants previously thought to be nulls retain BMP-independent functions (Eivers, 2009).
There were previous indications of a role for Dpp during segmentation in the Drosophila literature. It has been noted that in hypomorphic mutations of the BMP antagonist short gastrulation (sog, the homolog of Chordin), four copies of Dpp caused loss of some denticles and an increase in naked cuticle. In addition, it has been reported that in Dpp null mutants the posterior spiracles are replaced by an ectopic denticle belt. As noted in this study, Dpp nulls can present denticle belt fusions, a phenotype that has been observed previously in embryos injected with noggin mRNA. Dpp null phenotypes and those of Mad10 and Mad12 mutants (which lose C- terminal, but not linker phosphorylations) are not identical to those of Mad RNAi. It is suggested that this is because Mad also has Dpp-independent functions. Dissecting which effects of Mad are Dpp-dependent and which ones are independent will be an interesting area of future investigation. This study presents evidence for both taking place (Eivers, 2009).
Future work will have to address the level at which Mad regulation by MAPK and GSK3 interacts with other intracellular components of the Wg transduction pathway that result in similar phenotypes. The phenotypes observed for Mad loss-of function and mad phosphorylation-resistant linker mutants overexpression were very similar to those found for the armadillo/β-catenin, pangolin/lef1, legless/bcl9 and pygopus genes. The present study does not resolve the issue of whether the stabilized forms of Mad interact directly at the protein-protein binding level, thus modifying the core Wg pathway, or at level of DNA enhancers. Wnt responsive enhancers frequently contain Smad binding sites near TCF/Pangolin binding sites. In the vertebrates, direct binding between Lef1/Tcf and Smads 1 to 4 at the level of enhancer binding sites has been known for some time. What this study now shows is that Mad is also directly regulated at the level of its phosphorylation at GSK3 sites by Wg signaling. The possibility that Wg-stabilized Mad may bind to Armadillo/β-catenin, Pangolin/lef1, Legless/bcl9 and Pygopus independently of nearby Mad binding sites cannot be excluded at present. Mechanistic studies will have to explain the remarkable similarities between the stabilized Mad phenotypes and those of canonical Wg phenotypes in wing discs and bristles, segments and in neurogenic ectoderm in Drosophila, which suggest a widespread requirement for Mad in Wg signaling. Another aspect that will need to be addressed is why in Xenopus Wnt signaling through Smad1 has a complete requirement for β-Catenin, and in Drosophila the MGM can induce senseless only in regions in which β-catenin is also stabilized (Eivers, 2009).
Many developmental mechanisms have been conserved during evolution, but segmentation is one in which commonalities between Drosophila and the vertebrates have not been found. Segmentation in vertebrates relies on the cyclic oscillation of Notch pathway transcripts in the posterior paraxial mesoderm. In theory, Smad1/5/8 could provide an attractive regulator of the segmentation clock, because BMP signals have a duration of 1-2 hours in cultured cells, which can be extended by inhibiting GSK3. Wnt pathway genes cycle rhythmically in vertebrates, offering an interesting possibility for regulating Smad5/8 activity. Notch is required for segmentation in spiders, but not in Drosophila (Damen, 2007). Recently, it has been found that in the cockroach, an insect in which the segments are formed sequentially in a posterior growth zone (and not simultaneously as in Drosophila), stripes of Delta and Hairy mRNA (two genes of the Notch pathway) cycle rhythmically as in the vertebrates (Damen, 2007). This study has now found that Smad5/8 is required for the formation of segmental boundaries in Xenopus somites and that Mad is required for Drosophila segment patterning. However, the results do not establish whether similar molecular steps are affected in both organisms. The conservation of this unexpected conserved role for Mad/Smad is important from an Evo-Devo perspective because it suggests that the last common ancestor shared between Drosophila and vertebrates, Urbilateria, might have been segmented (Eivers, 2009).
These studies on Drosophila Mad have uncovered an unexpected role for Mad in the Wg signaling pathway. Mad/Smads are transcription factors that have low binding affinity for DNA and require other DNA binding proteins as co-factors in order to recognize the promoters and enhancers of hundreds of target genes. Future work will have to address how Mad or its partner Medea/Smad4 interact with proteins such as Armadillo/β-Catenin and Pangolin/Lef1 on Wnt-responsive promoters in Drosophila. The present study shows that Mad is required for Wg to signal, through its GSK3 phosphorylation sites, in a number of different in vivo assays. These include wing margin formation, sensory bristle induction in the wing, induction of the Wg induced gene senseless, the repression of neurogenic ectoderm, and segmental patterning. It is proposed that Mad serves as an integrator of patterning signals, which determine embryonic positional information. The finding that three major signaling pathways - MAPK, Wnt/GSK3 and BMP - are integrated at the level of Mad/Smad1/5/8 both in Drosophila and in the vertebrates has interesting implications for the evolution of animal forms through variations on an ancestral gene tool-kit (Eivers, 2009).
Grainy head (GRH) is a key transcription factor responsible for epidermal barrier formation and repair, whose function is highly conserved across diverse animal species. However, it is not known how GRH function is reactivated to repair differentiated epidermal barriers after wounding. This study shows that GRH is directly regulated by extracellular signal-regulated kinase (ERK) phosphorylation, which is required for wound-dependent expression of GRH target genes in epidermal cells. Serine 91 is the principal residue in GRH that is phosphorylated by ERK. Although mutations of the ERK phosphorylation sites in GRH do not impair its DNA binding function, the ERK sites in GRH are required to activate Dopa decarboxylase (Ddc) and misshapen (msn) epidermal wound enhancers as well as functional regeneration of an epidermal barrier upon wounding. This result indicates that the phosphorylation sites are essential for damaged epidermal barrier repair. However, GRH with mutant ERK phosphorylation sites can still promote barrier formation during embryonic epidermal development, suggesting that ERK sites are dispensable for the GRH function in establishing epidermal barrier integrity. These results provide mechanistic insight into how tissue repair can be initiated by posttranslational modification of a key transcription factor that normally mediates the developmental generation of that tissue (Kim, 2011).
Interestingly, putative ERK phosphorylation sites are also found in the N-terminal domain of Grhl3, a mammalian homolog of GRH. Given that grhl3 mutant mice display defects in both developmental skin barrier formation and wound-induced repair, and that ERK is required for mammalian wound repair, it is plausible that ERK phosphorylation of GRH might be an evolutionarily conserved event in animal epidermal wound repair. Drosophila GRH and mammalian Grhl proteins do not share extended blocks of amino acid sequence homology that include S-P or T-P motifs that are characteristic of ERK consensus sites, but functional phosphorylation sites can show rapid sequence drift during evolution (Kim, 2011).
Mutations of ERK phosphorylation site residues in GRH do not detectably influence its affinity to DNA binding sites in vitro, but the phosphorylation sites are required for GRH functional activity on epidermal wound enhancers in late embryonic epidermal cells. The wound-specific activity of GRH does not appear to involve phosphorylation-dependent nuclear localization, because the GRH protein is constitutively nuclear, either when expressed by endogenous promoters or epidermal GAL4 drivers. In addition, as measured by the strength of overexpressed ERK-site mutants of GRH in activating Ddc expression, the developmental transcriptional activation function of GRH was not significantly altered by the mutation of ERK phosphorylation sites. However, ERK phosphorylation of GRH-binding affinity might enhance its binding to a coactivator or prevent its binding to a corepressor that is specific during wound response. Because phosphomimetic GRH 2E expression did not constitutively activate transcription of GRH target genes in both developmental and wound response contexts, the phosphorylation of GRH alone is not likely to be sufficient for triggering activation of the wound response genes. Given that both GRH and FOS-D are required for the induction of Ddc and msn, and that FOS function can be also activated by ERK phosphorylation, it is believed that wound-induced signaling input through FOS or other transcription factors is also necessary for the transcription of these wound response genes along with the phosphorylation of GRH. Therefore, the ERK-dependent phosphorylation of both GRH and FOS upon injury may activate both transcription factors to synergistically induce many target genes that facilitate epidermal barrier regeneration (Kim, 2011).
Although GRH function in Drosophila embryonic epidermis is critical for both developmental generation and wound-triggered regeneration of epidermal barriers, it was not known whether it is required for the process of reepithelialization (epidermal wound closure) after wounding. Mammalian Grhl3 has been shown to be required for the keratinocyte wound closure in tissue culture, mainly through its activation of RhoGEF19. However, in Drosophila, it was found that the wound closure phenotypes between wild-type and grhIM homozygous embryos were indistinguishable, indicating that Drosophila GRH is not critical for reepithelialization after wounding (Kim, 2011).
Given the importance of the ERK-GRH axis in transcriptional activation of epidermal wound response genes, the signals and receptors upstream of ERK are of great interest. Mammalian cell culture studies suggest that receptor tyrosine kinases (RTK) are responsible for the wound-dependent activation of ERK. In Drosophila, stitcher (stit) encodes a Ret-family tyrosine kinase that contributes to transmission of an epidermal wound signal, because null mutations in stit result in a partial inhibition of wound-induced ERK phosphorylation, and reduced activation of wound enhancers in embryonic epidermal cells (Wang, 2009). It is very probable that another RTK(s) are responsible for the activation of ERK after epidermal wounding observed in stit null mutant embryos. The PVR RTK is a good candidate because its function has been shown to be required for wound healing in larval epidermal tissue (Kim, 2011).
The RTK-mediated activation of ERK-GRH axis appears to mediate other biological roles in addition to its role in embryonic epidermal barrier repair. GRH has been reported to mediate Torso RTK-dependent repression of the tailless gene in early embryogenesis. Therefore, although the ERK-GRH axis is dispensable for late embryonic epidermal barrier development, it appears to function in other developmental contexts. These distinct functions presumably depend on the context of different transcriptional enhancers with different transcription factor codes (Kim, 2011).
More importantly, the current findings suggest an important mechanism that may underlie injury-induced tissue regeneration. After wounding or amputation, developmental programming must be reinitiated to recover the original structure of the damaged tissue. In the context of the epidermis, GRH is a key transcription factor for generating a normal epidermal barrier during development in a manner independent of ERK phosphorylation. However, GRH is also persistently expressed in terminally differentiated epidermal cells of the embryo, larvae, and adult, and its function in barrier repair must be rapidly and robustly reactivated after wounding. In the current model, a semidormant state of GRH can be overcome by ERK phosphorylation to regain its ability to transcriptionally activate target genes like Ddc, msn, and many others that regenerate epidermal barriers. This model may also apply to c-Jun in mammals, which does not require JNK-dependent phosphorylation sites for developmental eyelid and neural tube closure, but does require those sites for closure of epidermal wounds. Thus, some regeneration processes in tissues or organs of diverse animal species after injury may be initiated through a similar molecular mechanism-posttranslational reactivation of essential transcription factors that are normally involved in developmental morphogenesis (Kim, 2011).
Constitutive heterochromatic regions of chromosomes are those that remain condensed through most or all of the cell cycle. In Drosophila, the constitutive heterochromatic regions, located around the centromere, contain a number of gene loci, but at a much lower density than euchromatin. In the autosomal heterochromatin, the gene loci appear to be unique sequence genes interspersed among blocks of highly repeated sequences. Euchromatic genes do not function well when brought into the vicinity of heterochromatin (position-effect variegation). Do blocks of centromeric heterochromatin provide an environment essential for heterochromatic gene function? To assay directly the functional requirement of autosomal heterochromatic genes to reside in heterochromatin, the rolled gene, which is normally located deep in chromosome 2R heterochromatin, was relocated within small blocks of heterochromatin to a variety of euchromatic positions by successive series of chromosomal rearrangements. The function of the rl gene is severely affected in rearrangements in which the rl gene is isolated in a small block of heterochromatin, and these position effects can be reverted by rearrangements which bring the rl gene closer to any large block of autosomal or X chromosome heterochromatin. There is some evidence that five other 2R heterochromatic genes are also affected among these rearrangements. These findings demonstrate that the heterochromatic genes, in contrast to euchromatic genes whose function is inhibited by relocation to heterochromatin, require proximity to heterochromatin to function properly; they argue strongly that a major function of the highly repeated satellite DNA, which comprises most of the heterochromatin, is to provide this heterochromatic environment (Eberl, 1993).
Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. This study shows that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. These results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, differences in the modes of Capicua downregulation by Torso and EGFR signaling are described, raising the possibility that such differences contribute to the tissue specificity of both signals (Astigarraga, 2007).
Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. This study links receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, this study shows that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. The sallimus/D-titin (sls) gene was identified as a novel muscle target of HOW-mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, it was demonstrated that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, these results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, these findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles (Nir, 2012).
Cellular differentiation during eumetazoan development is based on highly conserved signalling pathways. Two of them, the Notch and the EGFR signalling pathways, are closely intertwined. This study has identified two potential target sites of the Mitogen activated kinase (MAPK): the downstream effector kinase of EGFR and Hairless (H), the major antagonist of Notch signalling in Drosophila. Assuming that phosphorylation of these sites modulates H activity, a direct influence of EGFR signalling on Notch pathway regulation might be possible. This hypothesis was tested by generating a phospho-deficient and a phospho-mimetic H isoform and by assaying for their biological activity. The binding was assessed of known H interaction partners Su(H), Gro, CtBP and Pros26.4; this binding was similar between mutant and wild type H. Next eye, wing and bristle development, which are strongly affected by the overexpression of H due to the inhibition of Notch signalling, was assayed. Overexpression of the mutant constructs resulted in phenotypes similar to wildtype H overexpression, yet with subtle differences in phenotypic severity. However, large variations suggest that the mutated residues may be critical for the overall structure or stability of H. Albeit of minor impact, EGFR may fine tune Notch signalling via MAPK dependent phosphorylation of H (Nagel, 2014).
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