rolled/MAPK
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).
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 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).
During Drosophila oogenesis Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the MAPK cascade via the Drosophila EGF receptor (Egfr). Activation of this pathway induces different cell fates in the overlying follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially restricted and of constant intensity during mid-oogenesis. Gurken internalization can first be observed in all posterior follicle cells, abutting the oocyte from stage 4 to 6 of oogenesis. At the same time MAPK activation evolves in a spatially and temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms generated by target genes of the Egfr pathway (Peri, 1999).
In wildtype egg chambers rhomboid expression pattern can first be detected in a broad domain centered on the anterodorsal corner of the oocyte at the transition from stage 9 to stage 10a of oogenesis, at a time when MAPK activation cannot yet be detected in the follicular epithelium. Thereafter, RHO mRNA starts to be downregulated dorsally, beginning at the anterior margin of the domain. Dowregulation proceeds until the remnants of the first broad expression domain are reduced to a wide ring. rho expression is, at that time, retained only in a small patch in the dorsalmost cells and in two stripes extending laterally towards the nurse cell border. In these cells rho expression is then strongly upregulated and a refinement process begins that leads to a final pattern consisting of two L-shaped domains. Just as rho refinement starts in the dorsalmost part of the egg chamber, MAPK activation in the follicular epithelium first exceeds the detection threshold in exactly the same cells. During the following expansion of the MAPK domain, the strong activation at the leading edge seems to coincide with the refining stripes of rho expression. As the MAPK activation pattern reaches its final spectacle shape, again the dorsal and anterior portions of the MAPK staining pattern are associated with rho expression. In conclusion, rho expression precedes the detection of MAPK activation and both staining patterns are spatially and temporally well correlated. Rhomboid thus may be one of the factors responsible for generating the amplification and modulation of the initial Gurken signal (Peri, 1999).
Gurken signaling also induces the expression of a negative element in the Egfr pathway. Argos expression comes too late to explain the dynamic evolution of the MAPK and rhomboid patterns during stage 10 of oogenesis, but sprouty is expressed early enough to be part of the regulatory network controlling the Egfr pathway activation at stage 10. sprouty is induced in all posterior follicle cells abutting the oocyte at stage 4 to 6 of oogenesis. sty is absent from egg chambers lacking Gurken function, and thus sty may be one factor required to counteract the potential rho-dependent autoactivation of the Egfr pathway (Peri, 1999).
How does MAPK activation influence the morphogenesis of the developing egg shell? The gene Broad-complex is expressed and required in the anlagen of the dorsal respiratory appendages. It has been suggested, therefore, that Br-C acts as a marker specifying appendage fate. Gurken signaling leads to a repression of Br-C in the dorsalmost cells of the follicular epithelim. Br-C expression disappears in a sharply delineated dorsal-anterior patch coincident with the first observable MAPK activation. Later, when the MAPK activation refines to its distinct 'spectacle shape', Br-C expression is confined to two lateral domains showing weak MAPK activation surrounded by rings strongly staining for activated MAPK. Based on the striking complementarity of these patterns, it is proposed that at this stage Br-C is repressed by high levels and activated by low levels of MAPK activation. In addition, Br-C expression does not by itself specify dorsal appendage fate, as it is visible in duplicated anterior ringlike domains in completely ventralized ovaries that do not possess appendages. This adds a caveat to using Br-C expression as a fate marker for dorsoventral positions. It is proposed that Br-C instead is a general marker expressed in cells that undergo morphogenetic changes, consistent with its expression in the embryo and during earlier stages of oogenesis. It is proposed that Gurken initiates secondary processses in the follicular epithelium that modulate and amplify the initial activation of the Egfr pathway (Peri, 1999).
During Drosophila development Fos acts downstream from the JNK pathway. It can also mediate ERK signaling in wing vein formation and photoreceptor differentiation. Drosophila JNK and ERK phosphorylate D-Fos with overlapping, but distinct, patterns. Analysis of flies expressing phosphorylation site point mutants of D-Fos reveals that the transcription factor responds differentially to JNK and ERK signals. Mutations in the phosphorylation sites for JNK interfere specifically with the biological effects of JNK activation, whereas mutations in ERK phosphorylation sites affect responses to the EGF receptor-Ras-ERK pathway. These results indicate that the distinction between ERK and JNK signals can be made at the level of D-Fos, and that different pathway-specific phosphorylated forms of the protein can elicit different responses (Ciapponi, 2001).
The loss of wing vein tissue on expression of D-FosbZIP resembles phenotypes resulting from defects in the Drosophila epidermal growth factor receptor (DER) pathway, caused, for example, by loss-of-function alleles of DER itself or of other genes required for DER signaling, such as rhomboid, vein, ras, and ERK/rolled. Therefore, the above results might indicate that D-Fos acts as a mediator of the DER/ERK signaling pathway during wing vein differentiation. The artificial activation of this RTK pathway, by gain-of-function alleles or by overexpression of downstream effectors, gives rise to ectopic veins in the wing. To establish whether D-Fos might act epistatically to DER, and whether D-FosbZIP or a reduced D-fos gene dose (kayak alleles) might suppress such a phenotype was examined (Ciapponi, 2001).
The EllipseB1 (ElpB1) allele of DER represents an activated component of the DER/ERK signaling pathway. In addition to other phenotypes, for example, in the eye, ElpB1 animals consistently develop wings with ectopic wing vein material. Strikingly, both D-FosbZIP expression in the wing imaginal disc (using the 32B Gal4 driver) or the removal of one copy of D-fos in a kay heterozygote, suppresses this phenotype almost completely. To confirm that the observed effect is specific and caused by a reduction of endogenous D-Fos function, add-back experiments were performed in which this reduction was compensated by supplying extra wild-type D-Fos from a transgene, driven by the heat shock promoter (hs D-fos. Significantly, the presence of the D-fos transgene abrogates the suppression of ElpB1 by kay and reinstates the extra vein phenotype caused by elevated DER activity. This result confirms that the suppression of the activated DER allele is due to a loss of D-fos activity. Hence, D-Fos mediates wing vein patterning downstream from or in parallel with DER (Ciapponi, 2001).
Next, whether D-Fos mediates ERK signaling also during eye morphogenesis was investigated. Defects in photoreceptor differentiation can be induced by the RTK gain-of-function alleles ElpB1 and sevS11. The ElpB1 allele dominantly causes an abnormal eye phenotype that manifests itself in roughness and the occasional lack of outer photoreceptors. This phenotype can be suppressed largely by the removal of one copy of D-fos and restored subsequently by simultaneous transgenic expression of wild-type D-Fos. A gain-of-function transgene of the RTK-coding gene sevenless (sevS11) causes the characteristic appearance of ectopic R7 photoreceptor cells in nearly all ommatidia. The sevS11 phenotype can be suppressed by the expression of dominant-negative Fos. In flies carrying sevS11 in a heterozygous kay2 background, the ectopic R7 photoreceptor phenotype is suppressed significantly; the number of normal ommatidia increases from 5% to approximately 20%. Reintroduction of D-fos by a transgene in this double mutant background restores the percentage of ommatidia with extra photoreceptors observed in sevS11 heterozygous animals. Taken together, these results indicate that D-Fos can act as a rate-limiting component downstream from the RTKs Sev and DER during eye development (Ciapponi, 2001).
Considering that D-Fos is a transcription factor and based on the precedents of D-Jun and c-Fos, the most obvious role for D-Fos in DER and Sev signal transduction would be that of an effector of the Drosophila MAP kinase Rolled. Therefore, the effect of reducing D-fos activity in animals expressing the gain-of-function allele rlSem was examined. Expression of RlSem under UAS control in the wing imaginal disc results in an extra-vein phenotype, markedly when the flies are reared at 25°C and milder at 18°C. Simultaneous expression of D-FosbZIP along with RlSem causes a striking suppression of ectopic vein formation, whereas additional expression of full-length D-Fos leads to a strong enhancement of the phenotype. To confirm that the observed suppression of the rlSem phenotype was not due to an effect of D-Fos on the transgene promoters, a similar genetic interaction experiment was performed using the endogenous rlSem gain-of-function allele and kay2 allele. kay2 heterozygosity suppresses the rlSem-induced extra-vein phenotype. This effect is reverted by ubiquitous expression of D-Fos, indicating that the suppression is due specifically to the decreased activity of D-Fos. These genetic interactions confirm that the role of D-Fos in RTK signal is that of an effector of Rolled (Ciapponi, 2001).
The results described above reveal D-Fos as a downstream component of the ERK signal transduction pathway, yet previous genetic analyses have shown that the transcription factor serves as an effector of JNK. This raises the question of whether the function of D-Fos as recipient of ERK or JNK is mutually exclusive and determined by the cellular context, or whether the transcription factor may mediate both JNK and ERK responses in one tissue or one cell. The developing eye provides a system to approach such a question. Biochemical and genetic studies have indicated that the planar polarity pathway downstream from Frizzled (Fz) and Dishevelled (Dsh) leads to the activation of a JNK-type MAPK module. During retinal morphogenesis, this pathway controls the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline. Thus, in the developing eye the activity of JNK and ERK signal transduction can be monitored separately in vivo (by planar polarity and R-cell recruitment, respectively) (Ciapponi, 2001).
To determine whether D-Fos is involved in planar polarity signaling, the effect of D-FosbZIP expressed under the control of Gal4 drivers in the developing eye was examined. When D-Fos function is thus reduced, a striking combined phenocopy of defects in ERK and JNK signal transduction ensues. Sections of eyes of the hairy Gal4/UAS D-fosbZIP or of the sev Gal4/UAS D-fosbZIP genotypes display both a lack of photoreceptor cells, diagnostic of inadequate ERK signal transduction, and misoriented ommatidia, indicating defects in planar polarity signaling. This mutant phenotype makes it plausible that D-Fos, in addition to its role in photoreceptor cell recruitment downstream from ERK, acts as an effector of JNK signaling in planar polarity determination. Therefore, D-Fos mediates both JNK and ERK responses in a defined group of cells of the developing retina (Ciapponi, 2001).
To investigate the potential regulation of D-Fos by protein phosphorylation, in vitro kinase assays were performed in which recombinant JNK/Bsk and ERK/Rl were used as kinases and different bacterially expressed versions of D-Fos were used as substrates. In this in vitro setting, both Bsk and Rl could phosphorylate full-length D-Fos. To get an initial indication as to which residues might serve as target sites for ERK or/and JNK, the D-Fos amino acid sequence was compared with that of mammalian Jun and Fos proteins. The sequence alignments identified several sequences in D-Fos with similarity to confirmed JNK or ERK phosphorylation sites in the mammalian molecules. T89 and T93 of D-Fos correspond in their sequence context and relative location to established JNK phosphorylation sites in c-Jun. Alignment of the C-terminal parts of D-Fos and c-Fos show a conserved residue (T584) that corresponds to a previously described MAPK phosphorylation site in c-Fos. Moreover, several serine or threonine residues were identified that might serve as target sites for the proline-directed MAPKs. Mutant derivatives of D-Fos were generated in which one or more of these candidate phosphorylation sites was substituted by alanine. JNK/Bsk, but not ERK/Rl, can efficiently phosphorylate a fragment spanning the 170 N-terminal amino acids of D-Fos. When alanine substitutions are introduced in positions T89 and T93, the N-terminal D-Fos fragment is no longer an efficient substrate for JNK phosphorylation (Ciapponi, 2001).
Additional N-terminal residues that conform to the S/TP consensus (T234, S235, T237, and T254) are phosphorylated by neither JNK nor ERK. Similar to the 170-amino-acid N-terminal fragment, a D-Fos fragment covering the N-terminal 285 amino acids is not a substrate for ERK. This indicates that the N-terminal part of D-Fos is a good substrate for JNK/Bsk but not for ERK/Rl, and that the residues T89 and T93 serve as the main N-terminal JNK target sites (Ciapponi, 2001).
Interestingly, a small deletion that removes a sequence with remote similarity to the c-Jun delta-domain (amino acids 28-56), but that does not span the phosphorylation sites T89 and T93, completely abrogates phosphorylation of the D-Fos N-terminal fragment by JNK/Bsk. It is possible that this deletion destroys a delta-domain-like JNK docking site present in D-Fos (Ciapponi, 2001).
Next, the C-terminal part of D-Fos was analyzed; it contains seven potential phosphorylation sites for MAPKs. A fragment spanning the C-terminal 280 amino acids of D-Fos is, in contrast to the N terminus, phosphorylated efficiently by both Bsk and Rl. Alanine substitutions in all seven putative MAPKs target sites causes complete loss of phosphorylation. However, mutating subsets of the seven putative phosphorylation sites does not result in a complete loss of phosphorylation by either Rl or Bsk, indicating the presence of multiple phosphorylation sites in the C-terminal part of D-Fos. These results indicate that, at least in vitro, D-Fos is a direct substrate of both Drosophila JNK and ERK and that it contains overlapping, but distinct, sets of phosphorylation sites for the two kinases (Ciapponi, 2001).
After establishing which residues serve as substrates for JNK and/or ERK in vitro, it was important to determine the regulatory relevance of these sites in vivo. Transgenic fly strains expressing mutant forms of full-length D-Fos were generated in which either the putative N-terminal, JNK-specific phosphorylation sites, or the C-terminally located ERK and JNK substrate sites were replaced by alanine (D-FosN-Ala and D-FosC-Ala. In the D-Fospan Ala mutant, all the putative MAPK phosphorylation sites, both N- and C-terminal, were substituted by alanine. When the nonphosphorylatable D-Fospan Ala was expressed from a UAS-driven transgene under the control of the epidermal driver 69B Gal4, it gave rise to a strong thoracic cleft at the dorsal midline, resembling kay or hep mutants, or phenotypes that result from D-FosbZIP expression. Evidently, D-Fospan Ala represents a dominant-negative mutant that can interfere with JNK-dependent thorax closure (Ciapponi, 2001).
The relevance was investigated of subgroups of the D-Fos MAPK phosphorylation sites during thorax closure. Expression of D-FosN-Ala results in a distinctive thorax cleft, although not quite as pronounced as in the case of D-Fospan Ala. Importantly, this result shows that the N-terminal, JNK-specific phsosphorylation sites in D-Fos are required for a well-defined JNK-dependent developmental mechanism. Expression of D-FosC-Ala, which lacks the C-terminal phosphorylation sites, or of D-Foswt has no discernible effect. These results indicate that either the C-terminal sites play only an ancillary role and are not essential for JNK signaling in the signal transduction pathway controlling thorax, or that this mutant does not compete well with endogenous D-Fos and therefore has no dominant-negative effect in this context (Ciapponi, 2001).
Next, whether expression of the different phosphorylation mutants of D-Fos might also interfere with ERK responses was investigated. Expression of D-Fospan Ala in the posterior compartment of wing imaginal disc (using en Gal4) causes loss of wing vein material typical of mutants defective in DER to Rolled signaling. Thus, consistent with the observation that D-Fospan Ala has lost all substrate sites for both Rolled and Bsk, it acts as a dominant-negative form that interferes with D-Fos function in both the ERK and the JNK pathways (Ciapponi, 2001).
Interestingly, however, the D-FosN-Ala and D-FosC-Ala mutants influence the ERK and JNK response differently. D-FosN-Ala, which interfers dominantly with JNK-mediated thorax closure, has no effect on ERK-dependent wing vein formation. This is consistent with these sites not being substrates for Rolled. The D-FosC-Ala mutant, however, which is neutral in thorax development, causes loss-of-vein phenotype (Ciapponi, 2001).
To examine whether differential phosphorylation of D-Fos might also be used in the developing eye to distinguish between ERK and JNK signaling, the effect of the D-FosAla mutants on ERK-dependent photoreceptor cell recruitment and JNK-mediated ommatidial rotation was examined. Different D-FosAla mutants were expressed along with sevS11 in the eye imaginal disc under the control of the sevenless enhancer. As in the case of wing vein formation, D-FosN-Ala does not alter the sevS11 phenotype, whereas the expression of D-FosC-Ala or of D-Fospan Ala causes a significant suppression of the extra R7-cell recruitment. Thus, ERK signaling, whether it is triggered by DER or by Sev, appears to require only the C-terminal phosphorylation sites of D-Fos (Ciapponi, 2001).
Next, whether the D-FosAla mutants could suppress the ommatidial misrotation phenotype that is elicited by overexpression of Fz and the ensuing activation of JNK was tested. Coexpression of all Ala mutants and Fz under the control of sev Gal4 leads to a significant suppression of the misrotation phenotype observed in flies expressing Fz alone. Wild-type D-Fos does not have this effect. These results indicate that the JNK-phosphorylation sites of D-Fos are required for the manifestation of the Fz gain-of-function phenotype. The Fz-JNK response in the eye is also affected by mutation of the C-terminal sites that do not visibly disturb the thorax closure response. This might be explained by the higher sensitivity of the ommatidial rotation paradigm or a higher relative expression of the FosAla mutant transgene in the photoreceptor cells (Ciapponi, 2001).
The dominant phenotypic effects of D-FosAla expression support the interpretation that Fos is regulated by protein phosphorylation to mediate developmental decisions and indicates that the residues identified by mutagenesis and in vitro kinase assay are required for in vivo function. Moreover, these findings indicate clearly that the function of D-Fos as a mediator of JNK/Bsk and ERK/Rl cascades is in both cases that of a direct kinase substrate (Ciapponi, 2001).
An important question raised by the finding that D-Fos can mediate signaling by both ERK and JNK is how the decision between these distinct cellular responses is made, that is, how the cell 'knows' which program to execute when D-Fos becomes phosphorylated. Several mechanisms have been suggested to contribute to signal specificity in such situations, in which one protein mediates different cellular responses. One model proposes a combinatorial mechanism by which several factors with overlapping broad responsiveness have to cooperate to mediate a defined specific cellular behavior. However, the observation that D-Fos participates in ERK as well as JNK signal transduction in the same group of cells of the developing Drosophila eye, by regulating photoreceptor differentiation and ommatidial rotation, respectively, argues against cell type-specific cofactors that modulate the response to D-Fos phosphorylation. The distinct substrate sites in D-Fos phosphorylated by ERK and JNK raise a novel possibility to explain the signal-specific D-Fos response. It is suggested that D-Fos exists in two different activated forms, depending on whether it is phosphorylated by ERK or JNK. These differentially phosphorylated forms might then selectively trigger either the ERK or the JNK response. This idea is supported by in vivo experiments in which phosphorylation site-specific point mutants of D-Fos were expressed in the developing fly. A mutant that lacks all phosphorylation sites interferes dominantly with both ERK and JNK signaling in thorax closure, the wing, and the eye imaginal disc, supporting further the general relevance of D-Fos phosphorylation in developmental decisions. D-Fos mutants lacking subsets of phosphorylation sites, however, affected JNK and ERK signal responses differentially. An N-terminal cluster of JNK sites that is not phosphorylated by ERK is critical for the JNK response in vivo. A mutant lacking these sites interfers with thorax closure and planar polarity regulations, both bona fide JNK responses, but not with wing vein formation or photoreceptor differentiation, which are regulated by ERK. Conversely, a mutant that removes all ERK substrate sites dominantly suppresses processes normally controlled by this MAP kinase. These data indicate that signal-responsive transcription factors, such as D-Fos, may have different signal-specific functions. It is tempting to speculate that such a mechanism might be used by other signaling proteins that are receptive to different upstream signals (Ciapponi, 2001).
The Heartless (Htl) FGF receptor is required for the differentiation of a variety of mesodermal tissues in the Drosophila embryo, yet its ligand is not known. Two FGF genes, thisbe (ths; FGF8-like1) and pyramus (pyr; FGF8-like2), have been identified which probably encode the elusive ligands for this receptor. The two genes were named for the 'heartbroken' lovers described in Ovid's Metamorphoses because the genes are linked and the mutant phenotype exhibits a lack of heart. The genes exhibit dynamic patterns of expression in epithelial tissues adjacent to Htl-expressing mesoderm derivatives, including the neurogenic ectoderm, stomadeum, and hindgut. Embryos that lack ths+ and pyr+ exhibit defects related to those seen in htl mutants, including delayed mesodermal migration during gastrulation and a loss of cardiac tissues and hindgut musculature. The misexpression of Ths in wild-type and mutant embryos suggests that FGF signaling is required for both cell migration and the transcriptional induction of cardiac gene expression. The characterization of htl and ths regulatory DNAs indicates that high levels of the maternal Dorsal gradient directly activates htl expression, whereas low levels activate ths. It is therefore possible to describe FGF signaling and other aspects of gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004; Gryzik, 2004).
The fact that FGF8-like1 and FGF8-like2 are expressed in the ectoderm and are required for cell shape changes of mesoderm cells indicates a non-cell-autonomous function of FGF8-like1 and FGF8-like2. However, the FGF-receptor Htl is specifically expressed in the mesoderm cells. In order to test whether FGF8-like1 and FGF8-like2 are required for the activity of Htl in the mesoderm, the activation of the downstream signaling component MAP kinase was measured by using an antibody that recognizes the activated double-phosphorylated form of MAP kinase. In the wild-type, activated MAP kinase can be detected in the leading-edge cells of the migrating mesoderm. This early activation of MAP kinase in the mesoderm depends on the presence of Htl and its downstream signaling factor Dof. To test whether FGF8-like1 and FGF8-like2 are required for activation of MAP kinase in the mesoderm cells during migration, embryos homozygously mutant for Df(2R)ED2238 or Df(2R)ED2230 were stained with the dpERK antibody. Strikingly, only embryos mutant for Df(2R)ED2238 failed to exhibit dpERK staining in the mesoderm, whereas Df(2R)ED2230 mutant embryos looked like the wild-type. The defect in MAP kinase activation in Df(2R)ED2238 mutant embryos is specific for Htl FGF receptor activation because the staining of other cells that activate the MAP kinase pathway via the EGF receptor remains unimpaired (Gryzik, 2004).
During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of Rolled, a mitogen activated protein tyrosine kinase. Key nuclear targets of Rolled are two
antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2),
an activator. A critical regulator of this process, Mae, can interact with both
Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae
derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the
mechanism of Pnt-P2 regulation by Mae remained undefined. This study finds
that efficient
phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional
docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to
Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2
activity. Docking site blocking provides a new mechanism whereby the cell can
precisely modulate kinase signaling at specific targets, providing another layer
of regulation beyond the more global changes effected by alterations in the
activity of the kinase itself (Qiao, 2005).
The findings in this work, combined with prior results, suggest how Yan, Pnt-P2 and Mae work together to determine cell fate in response to the activation of the
RTK pathways. In the absence of MAPK activation, unphosphorylated Yan polymers
outcompete Pnt-P2 for access to ETS-binding sites, creating a repressed state of
the target genes. Upon RTK activation, activated phospho-Rolled MAPK
enters the nucleus and phosphorylates a small amount
of the monomeric Yan. By binding to Yan and blocking polymer interactions, basal
levels of Mae likely help to maintain an appropriate concentration of free Yan
in the nucleus. Phosphorylation of Yan triggers its
cytoplasmic export with the help of CRM1. The decrease in
free Yan then drives the equilibrium away from the DNA-bound polymer. Meanwhile,
the antagonist of Yan, Pnt-P2, becomes activated by Rolled MAPK phosphorylation,
presumably through enhanced binding to transcriptional coactivators CREB binding
protein (CBP) and p300, which act to bridge the DNA-bound transcription factors
and the basal transcription complex. Since Mae is regulated by
Yan and Pnt-P2, inactivation of Yan and activation of
Pnt-P2 leads to increasing amounts of Mae and further removal of Yan repression.
These processes could easily lead to runaway expression of differentiation
genes. As revealed in this study, however, another job of Mae is to block the
MAPK/Rolled docking site on Pnt-P2, inhibiting Pnt-P2 phosphorylation, which in
turn attenuates transcriptional activity. This negative feedback loop ensures a
level of transcription appropriate for normal development (Qiao, 2005).
In Drosophila cells, phosphorylation of eIF4E (eukaryotic initiation factor 4E) is required for growth and development. In Drosophila, LK6 is the closest homologue of mammalian Mnk1 and Mnk2 [MAPK (mitogen-activated protein kinase) signal-integrating kinases 1 and 2 respectively] that phosphorylate mammalian eIF4E. Mnk1 is activated by both mitogen- and stress-activated signalling pathways [ERK (extracellular-signal-regulated kinase) and p38 MAPK], whereas Mnk2 contains a MAPK-binding motif that is selective for ERKs. LK6 possesses a binding motif similar to that in Mnk2. The present study shows that LK6 can phosphorylate eIF4E at the physiological site. LK6 activity is increased by the ERK signalling pathway and not by the stress-activated p38 MAPK signalling pathway. Consistent with this, LK6 binds ERK in mammalian cells, and this requires an intact binding motif. LK6 can bind to eIF4G in mammalian cells, and expression of LK6 increases the phosphorylation of the endogenous eIF4E. In Drosophila S2 Schneider cells, LK6 binds the ERK homologue Rolled, but not the p38 MAPK homologue. LK6 phosphorylates Drosophila eIF4E in vitro. The phosphorylation of endogenous eIF4E in Drosophila cells is increased by activation of the ERK pathway but not by arsenite, an activator of p38 MAPK. RNA interference directed against LK6 significantly decreases eIF4E phosphorylation in Drosophila cells. These results show that LK6 binds to ERK and is activated by ERK signalling and it is responsible for phosphorylating eIF4E in Drosophila (Parra-Palau, 2005; full text of article).
EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. This study found that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrates that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. These observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways (Reddy, 2013).
In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. This study addresses the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H)MAPK-ko and a phospho-mimetic Su(H)MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ and vgBE-lacZ). In general, Su(H)MAPK-ko induced a stronger response than wild-type Su(H), whereas the response to Su(H)MAPK-ac was very weak. Notch target genes cut, wingless and vgBE-lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DERact) or the MAPK (rlSEM) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones (Auer, 2015).
Continued: MAP kinase Protein Interactions part 3/3 | part 1/3
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