Gene name - rolled
Synonyms - MAP kinase
Cytological map position - h38--h41
Function - serine/threonine protein kinase
Symbol - rl
Genetic map position - 2-55.1
Classification - Map kinase - ERK
Cellular location - cytoplasmic and nuclear
rolled/MAP kinase is essential to the proper functioning of the Ras signaling pathway. Mammalian homologs are known as ERKs (extracellular signal-regulated kinases) because of their roles in transducing signals from outside the cell into the nucleus. Targets of Rolled in Drosophila include Pointed, Anterior open (commonly referred to as Yan), Seven in absentia (Sina) and Jun related antigen (Jun or DJun). For example, although induction of Sina is not regulated by Rolled, its biological activity is regulated by Ras pathway phosphorylation (Dickson, 1992). The same holds true for Anterior open. Anterior open activity is targeted by the Ras pathway (Gabay, 1996). Likewise, the Ras pathway activates Pointed protein (Brunner, 1994b) and Jun related antigen, commonly known as Jun (Peverali,1996).
The development of the Drosophila eye is controlled in part, by the Ras pathway. This provides a good illustration of rolled/Mapk functions. The Drosophila compound eye is made up of a cluster of single simplified eye elements, collectively termed ommatidia. Each ommatidium contains eight photoreceptors (R1-R8), differentiated during larval development in a process of discrete steps from the larval eye imaginal disc: in order of determination, they are R8, followed by the pairs R2/R5, R3/R4, and R1/R6, and finally R7. Induction of R7 involves signals from the R8 photoreceptor. The R8 photoreceptor presents on its surface a ligand, Bride of Sevenless, that binds and activates Sevenless receptor tyrosine kinase in the R7 precursor. Autophosphorylated Sevenless initiates a Ras1-mediated cascade, which eventually activates transcription factors in the nucleus via Raf1 and MAP kinases, resulting in R7 development (Yamamoto, 1994 and references).
The presence of functional R7 photoreceptor cells can be detected by a simple behavioral test. Given a choice between an ultraviolet (UV) and a visible light source, wild-type flies will move towards the UV light. Flies lacking R7 cells, however, will move towards the visible light. This behavior has been used to identify mutations in genes that prevent the development of R7 photoreceptor cells and has led to the identification of sevenless and bride of sevenless genes. This behavioral screen can be used to identify dominant mutations that result in the activation of the sevenless signal transduction pathway, even in the absence of its inducing signal, the Boss protein (Brunner, 1994a and references).
Sevenmaker, a gain of function rolled mutation, specifies R7 photoreceptor cells independently of boss and sev functions. In boss mutant flies, the R7 cell is missing, whereas in Sev gain of function mutants many ommatidia contain multiple R7-like cells (Brunner, 1994a). Rolled acts downstream of Ras1 and Raf in specifying the R7 fate. In addition, rolled plays a key role in the Torso pathway. Gain of function Sev results in the ectopic acivation of the Torso signaling pathway in a manner similar to its effect on the sevenless pathway in the developing eye. Loss-of-function mutations in rolled can suppress gain-of-function torso mutations. Thus Rolled acts in the terminal pathway responsible for specification of terminal structures immediately after fertilization. Likewise rolled mutations affect signaling of the Drosophila Epidermal growth factor receptor. Gain-of-function Sevenmaker mutations produce extra wing veins close to the wing margin. In addition, rolled mutations modify EGF-R signaling in the establishment of the dorsoventral polarity of the egg shell and the embryo (Brunner, 1994a).
Rolled activity is targeted during heat shock induced stress in cultured Drosophila cells. Heat shock activates a MAP kinase phosphatase, resulting in a reduction of Rolled activity over the course of a 15 minute heat shock regime (raising the temperature from 25 to 37 degrees) (Cornelius, 1995).
Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still
poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by
blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).
The reduction in midline glia (MG) cell number due to apoptosis, as well as the requirement of the RAS/MAPK pathway for MG survival, has been documented using various MG-specific enhancer trap lines and reporter fusion constructs. The MG are visualized using a reporter fusion construct for the slit gene (sli-lacZ) in which a 1 kb fragment of the slit promoter confers expression specifically to the MG. Using a ß-gal antibody to monitor the developmental profile of the MG, about ten cells per segment expressing sli-lacZ are detectable at midembryogenesis (stage 13). By the end of embryogenesis at stage 17, the number of sli-lacZ-positive cells is reduced to approximately three per segment. Since the sli-lacZ expression is specific for the MG, sli-lacZ-expressing cells are referred to as MG (Bergmann, 2002).
Prominent activation of MAPK has been identified in the MG cells, but its functional role has not been determined. The fate of the MG in mapk-deficient embryos has been analyzed. Compared to wild-type embryos, the initial generation of the MG appears to be normal. However, by stage 17 (the end of embryogenesis), none of the MG in mapk-deficient embryos survive. This finding suggests that MAPK is required for MG survival in wild-type embryos (Bergmann, 2002).
The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).
The mutant analysis revealed that MAPK is required to suppress the activity of HID for MG survival. If hid is mutant in mapk-deficient embryos (i.e., in mapk; hid double mutants), MAPK is no longer needed for the survival of the MG. Thus, MAPK-mediated survival of the MG functions through inhibition of HID (Bergmann, 2002).
In hid mutant embryos there is a 2-fold increase of the MG compared to wild-type. Approximately six MG per segment survive in hid embryos compared to the 2.8 MG per segment in wild-type, indicating that hid is genetically required for MG cell death. MAPK activity is required for MG survival. Does the level of MAPK activity determine the final number of surviving MG cells? Mutational activation of MAPK, using a dominant allele of MAPK termed Sevenmaker or mapkSem, promotes survival of extra MG. About 6.0 MG per segment survive in stage 17 mapkSem embryos, providing additional evidence that MAPK is required for MG survival. Remarkably, the number of surviving MG in mapkSem embryos is very similar to the number of surviving MG in hid mutant embryos. In both cases, approximately six MG survive per segment. Therefore, it was determined whether the six surviving MG in mapkSem embryos correspond to the same MG that survive in hid mutant embryos by double mutant analysis. Stage 17 mapkSem; hid double mutant embryos contain on average 6.6 MG, or slightly more than the single mutants alone. This result strongly suggests that hid expression and MAPK activation occur in largely the same set of MG, that is, in a group of about six MG. If MAPK activation and hid expression would occur in different MG independently of each other, then the mapkSem;hid double mutant would be expected to be the composite of the individual mutants and a total of about ten to twelve MG would survive in the double mutant, similar to what has been observed in H99 mutant embryos. It is inferred from the double mutant analysis that the survival of approximately six MG is regulated by MAPK-dependent inhibition of HID. As long as MAPK is activated, these MG survive (as seen in the activated mapkSem mutant). However, MG in this group that does not maintain activated MAPK are eliminated by HID-induced apoptosis. Thus, the coordinated expression of HID and activation of MAPK regulate the final MG cell number (Bergmann, 2002).
MAPK suppresses hid activity in two ways: via downregulation of its transcription and via phosphorylation of HID protein. However, hid mRNA and protein are readily detectable in the surviving MG of wild-type embryos. Therefore, transcriptional downregulation of hid does not account for MG survival. This prompted a test to see whether inhibitory phosphorylation of HID by MAPK might be critical for MG survival. For this purpose, advantage was taken of an observation that overexpression of HID in the MG using the MG-specific sli-GAL4 driver and UAS-hid transgenes is not sufficient to induce MG apoptosis. Even two copies of the UAS-hid transgenes are not able to ablate the MG. This is contrary to findings in other tissues in which expression of hid induces cell death very well. However, since MAPK is activated in the MG and required for MG survival, it was hypothesized that even overexpressed HID might be inactivated via MAPK phosphorylation (Bergmann, 2002).
To examine this further, UAS-hid transgenes were generated that alter the five phosphoacceptor residues of the MAPK phosphorylation sites to nonphosphorylatable Ala residues (UAS-hidAla5). The UAS-hidAla5 transgenes driven by sli-GAL4 induce apoptosis in the MG very efficiently. One copy of a UAS-hidAla5 transgene is sufficient for the ablation of the MG. Occasionally, some embryos are recovered in which the ablation of the MG is incomplete. However, nerve cord preparations reveal that in these embryos, only a small fraction of the MG survives compared to wild-type. Some segments completely lack MG cells, while others just contain one remaining MG. The MG is required for separation of the commissural axon tracts of the CNS. Consistently, expression of the UAS-hidAla5 transgenes and consequently ablation of the MG causes a fused commissure phenotype. In summary, this analysis demonstrates that MG survival requires suppression of HID activity by MAPK. The MAPK phosphorylation sites in HID are critical for this response, providing an important mechanism for the regulation of MG number (Bergmann, 2002).
Activation of MAPK usually requires activation of RAS, which in turn is activated by receptor tyrosine kinase (RTK) signaling: this demonstrates that MG survival depends on RAS, which is consistent with the model. Within the embryonic CNS, the Drosophila homolog of the Epidermal growth factor receptor (Egfr) is specifically expressed and required for MG differentiation. The requirement of Egfr signaling for MG survival was examined (Bergmann, 2002).
Due to severe developmental defects in egfr mutants, only a few MG start forming at stage 11, and none of them survive. Thus, it is difficult to directly study the requirement of the Egfr for MG survival. To overcome this problem, a dominant-negative mutant of the Egfr (UAS-EgfrDN) was expressed in the MG using the sli-GAL4 driver in otherwise wild-type embryos. In this way, Egfr activity is specifically diminished in the MG after their generation. As expected, the MG form normally in these embryos. However, most of the MG die during subsequent developmental stages and only a few survive to the end of embryogenesis, indicating a direct requirement of the Egfr for MG survival. To determine whether the MG death in this experimental condition is due to failure to inhibit HID, EgfrDN was expressed in the MG of hid mutants. In this genetic background, on average 6.1 MG cells survive, demonstrating that MG survival requires functional Egfr signaling to suppress HID activity (Bergmann, 2002).
The spi gene is required for MG survival and encodes a candidate trophic factor for MG survival. To prove that spi function is required to suppress hid activity, the fate of the MG in spi;hid double mutant embryos was determined. The MG survive in spi embryos if hid is removed as well, and it is concluded that the survival function of spi is mediated through suppression of hid-induced apoptosis (Bergmann, 2002).
The question arises as to which cells process mSPI and provide a source of sSPI for MG survival. Since spi is ubiquitously expressed, it is difficult to determine histochemically where sSPI, the active ligand, is generated. Therefore, a genetic approach was used; whether the loss of MG in spi mutant embryos can be rescued by expression of the membrane bound inactive precursor (UAS-mSPI) either in the MG (using the sim-GAL4 driver) or in neuronal axons (using the elav-GAL4 driver) was examined. It was reasoned that the MG would be rescued in spi mutant embryos only if mSPI is presented in the location where it is normally processed for MG survival in wild-type embryos. Presentation of mSPI by the MG itself does not result in rescue of the MG in spi mutant embryos, ruling out an autocrine mechanism. In contrast, expression of mSPI in neuronal axons appears to be sufficient for MG survival in spi embryos. This argues in favor of a paracrine mechanism. In control experiments, sSPI was examined using these two Gal4 drivers in wild-type embryos. With both GAL4 drivers an increase in the number of MG cells is detected, indicating that they are expressed at the right time and that the MG does not fail to secrete Spi once it has been processed (Bergmann, 2002).
A key regulator of Spi activation is rhomboid, a gene encoding a cell surface, seven-pass transmembrane protein that appears to function as a serine protease directly cleaving mSPI. rhomboid has been implicated in suppression of MG apoptosis. Ectopic expression of Rho in neurons (elav-Gal4/UAS-Rhomboid) promotes an excess of MG, suggesting that neurons have the capacity to process endogenous mSPI. Another essential protein for Spi processing is Star. Star mutants display an MG phenotype similar to spi. STAR regulates intracellular trafficking of mSPI. Expression of Star from the neurons but not from the MG rescues the Star phenotype in the MG. Thus, this analysis clearly demonstrates that the sSPI signal for MG survival is generated and secreted by neurons (Bergmann, 2002).
The surviving MG in late stage embryos are in close contact to commissural axons. In embryos lacking the commissureless (comm) gene, the commissural axons are absent. In comm embryos the MG die prematurely, and some survivors become misplaced laterally along the longitudinal axon tracts. The location of the MG along the longitudinal axons in comm mutant embryos as well as their close contact to commissural axons in wild-type embryos has prompted the suggestion that axon contact is required for MG survival. Axon contact appears to permit the MG to respond to trophic signaling, which is necessary for its survival. Consistent with this notion trophic signaling provided by sSPI/Egfr is present only in MG associated with longitudinal axons in comm;hid double mutants. Thus, it was asked whether axon contact-mediated Egfr signaling in the MG is required to activate MAPK, which in turn suppresses the cell death-inducing ability of HID (Bergmann, 2002).
To address this question, the fate of the MG was examined in comm mutant embryos which are at the same time mutant for hid (comm;hid double mutants) or carry the dominant active mapk allele, mapkSem (mapkSem;comm double mutants). Strikingly, a substantial number of the MG survive even in the absence of axonal contact if hid function is removed or if MAPK is activated. This strongly suggests that axon contact is necessary to suppress HID via MAPK. Only MG in proximity to neurons undergo Egfr signaling. The additional MG that survive along the midline in comm;hid mutants do not express spry, that is, do not receive an Egfr signal, and survive only because hid is absent in this experimental condition (Bergmann, 2002).
It is noted that active MAPK is capable of rescuing a total of six MG based on analysis of mapkSem embryos. Presumably, this MAPK activation in mapkSem embryos is inherited from the differentiation period of the MG. However, only three MG survive by stage 17 in wild-type embryos. It is proposed that of the group of six MG that require MAPK for survival, only the three surviving cells make adequate axon contacts necessary to receive sufficient quantities of the survival factor sSPI. According to this model, the remaining three MG die because they lose the competition for axon contact and do not receive levels of sSPI that are high enough to inactivate HID via phosphorylation by MAPK. If additional sSPI is provided in the midline, additional MG can be rescued. The limited availability of axon-derived sSPI would serve to match the number of MG to the length of commissural axons requiring ensheathment. Thus, the regulation of MG number and survival represents a genetically defined example of the classical trophic theory of cell survival (Bergmann, 2002).
The regulation of MG apoptosis in Drosophila bears striking overall similarity to the regulation of glial cell death in the rat optic nerve. There is an early dependence of the oligodendroglia in the rat optic nerve on growth factors for differentiation followed by a dependence on axon contact for survival. However, it is not clear how the oligodendroglia in the rat optic nerve survive upon axon contact. Since mammalian homologs for many of the components in the apoptotic pathway both upstream and downstream of Drosophila HID are known, it will be interesting to analyze whether similar molecules regulate apoptosis and cell number in the mammalian nervous system. Therefore, molecular genetic studies in Drosophila promise considerable insight for advancing an understanding of the basic control mechanisms involved in the regulation of apoptosis in the context of a developing organism in vivo (Bergmann, 2002).
The rolled locus is found in a heterochromatic region of chromosome 2 that is considered to remain condensed (and for the most part transcriptionally inactive) throughout all or most of the cell cycle. rolled lies in what is considered to be alpha heterochromatin, a chromosome region that makes up the chromocenter of polytene salivary gland chromosomes. The chromocenter is not thought to be polytenized, that is, it is not thought to undergo repeated rounds of DNA replication resulting in multiple copies of active genes. The chromocenter is thought to be made up of DNA and protein in a tightly knit dense structure of which is transcriptionally inactive. Such heterochomatic regions, which make up 30% of the Drosophila genome, have a much lower density of genes as compared to euchromatin.
rolled gene activity is unusual in that it requires the surrounding heterochromatin for gene function. rolled gene activity is severly impaired by bringing rolled close to any euchromatic position; however, these position effects can be reversed by chromosomal rearrangements that bring the rolled gene closer to any block of autosomal or X chromosome heterochromatin (Eberl, 1993). Heterochromatic rolled is extensively polytenized and transcriptionally active in salivary gland chromosomes. rolled undergoes polytenization in salivary gland chromosomes to a degree comparable to that of euchromatic genes, despite its deep heterochromatic location. Sequences on either side of rolled are severly underrepresented in polytene chromosomes and are considered to appear in alpha-heterochromatin. It is suggested that multiple domains of functionally active, complex DNA sequences, composed of single-copy genes and middle-repetitive elements, are present within the proximal heterochromatin of chromosome 2. It may well be that heterochromatin is not the transcriptional desert that it was once thought to be (Berghella, 1996).
Patterning of the terminal regions of the Drosophila embryo relies on the gradient of phosphorylated ERK/MAPK (dpERK), which is controlled by the localized activation of the Torso receptor tyrosine kinase. This model is supported by a large amount of data, but the gradient itself has never been quantified. This study presents the first measurements of the dpERK gradient and establishes a new intracellular layer of its regulation. Based on the quantitative analysis of the spatial pattern of dpERK in mutants with different levels of Torso as well as the dynamics of the wild-type dpERK pattern, it is proposed that the terminal-patterning gradient is controlled by a cascade of diffusion-trapping modules. A ligand-trapping mechanism establishes a sharply localized pattern of the Torso receptor occupancy on the surface of the embryo. Inside the syncytial embryo, nuclei play the role of traps that localize diffusible dpERK. It is argued that the length scale of the terminal-patterning gradient is determined mainly by the intracellular module (Coppey, 2008).
This study identifies the nuclear trapping of dpERK as a mechanism responsible for the intracellular spatial processing of the terminal signal. This conclusion is based on the analysis of the dynamics of the wild-type dpERK gradient. Between nuclear cycles 10 and 14, the dpERK levels are amplified at the termini and attenuated in the subterminal regions of the embryo. The observed dynamics of the dpERK gradient is consistent with a model where dpERK is a diffusible molecule, which is trapped and dephosphorylated by the nuclei. A uniform increase in the nuclear density would increase the trapping of the dpERK molecules at the poles and prevent their diffusion to the middle of the embryo. This is consistent with biochemical and imaging data showing that dpERK rapidly translocates to the nucleus, which can also serve as a compartment of dpERK dephosphorylation. In addition, the model makes a testable prediction about the dynamics of the nucleocytoplasmic (N/C) ratio of phosphorylated MAPK (Coppey, 2008).
The nuclear and cytoplasmic levels of dpERK were quantified in cycle 13 and 14 embryos. Plotting the nuclear and cytoplasmic profiles against each other gives a clear linear relationship, as predicted by a simple formula. Furthermore, the nucleocytoplasmic ratio clearly increases between these two nuclear cycles: the N/C ratio is ~1.4 and ~2 at nuclear cycles 13 and 14, respectively. These measurements show that the nuclear trapping rate is indeed an increasing function of the nuclear density, as predicted by the model (Coppey, 2008).
The observed N/C ratios show that a significant fraction of total dpERK nuclear. As a consequence, defects in the nuclear density should generate clear defects in the gradient. This can be tested in mutants with 'holes' in the nuclear density in blastoderm embryos. For example, in shakleton (shkl) embryos, the migration of nuclei to the poles is delayed and a number of embryos exhibit major disruptions in nuclear density. As predicted by the model, shkl embryos show striking disruptions in the dpERK gradient. The quantified posterior gradient of this particular mutant embryo shows a clear local correlation with the nuclear distribution, emphasizing the role of the nuclei at this stage. In early embryos, the gradient is more extended, presumably reflecting the lack of the nuclei at the poles. Similar defects were found in the giant nuclei (gnu) mutant embryos, which show a different type of defect in nuclear organization. These results support the model in which the syncytial nuclei play an important role in shaping the dpERK gradient (Coppey, 2008).
To summarize, it is proposed that the dpERK gradient is controlled by a cascade of at least two diffusion-trapping modules. In the extracellular compartment, a ligand-trapping mechanism, identified in previous studies, establishes a sharp gradient of Torso receptor occupancy. A similar mechanism regulates the dpERK gradient inside the embryo, where syncytial nuclei act as traps that localize diffusible dpERK. At this time, it cannot be ruled out that the observed sharpening of the dpERK gradient can be modulated also by changes in the spatial distribution of the Torso ligand, but currently there are no data in support of this mode of regulation (Coppey, 2008).
The dynamics of the dpERK gradient is qualitatively different from that of the Bicoid gradient, which remains stable during the last five nuclear divisions. It has been proposed that a stable gradient of Bicoid can be established in the absence of Bicoid degradation, because of the reversible trapping of Bicoid by an exponentially increasing number of nuclei. The differences between the dynamics of the Bicoid and dpERK gradients are attributed to two effects. The first effect is due to the differences in the 'chemistries' of the two systems: the morphogen in the terminal system is degraded (MAPK is dephosphorylated), whereas the anterior morphogen is stable (it is proposed that Bicoid is not degraded on time scale of the gradient formation). The second effect is due to the differences in the initial conditions: by the 10th nuclear division, which is the starting point of the activation of the terminal system, Bicoid gradient is essentially fully established. Thus, a common biophysical framework can describe the Bicoid and dpERK gradients. It remains to be determined whether the nuclear export affects the length scale of the Dorsal gradient, which patterns the dorsoventral axis of the embryo (Coppey, 2008)
There are several extended regions of amino acid identity with rat ERK1 and -2 and yeast FUS3 and KSS1. The sequence is most similar to rat ERK1 and -2 over its entire length (80% identity) (Biggs, 1992).
Rolled protein contains 11 conserved kinase subdomains characteristic of kinase proteins. The gain of function Sevenmaker mutation (see above: Biological overview ) is found in the C-terminal end of kinase domain XI, the putative adenosine triphospate-binding site is located in conserved domain II (Brunner, 1994a and references).
The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components (Canagarajah, 1997).
date revised: 30 March 2002Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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