EGF receptor
Egfr and the intracellular redox balance Modulation of reactive oxygen species (ROS) plays a key role in signal transduction pathways. Selenoproteins act controlling the redox balance of the cell. This study reports how the alteration of the redox balance caused by patufet (selDptuf), a null mutation in the Drosophila melanogaster selenophosphate synthetase 1 (sps1) gene, which codes for the SelD enzyme of the selenoprotein biosynthesis, affects the Ras/MAPK signaling pathway. The selDptuf mutation dominantly
suppresses the phenotypes in the eye and the wing caused by hyperactivation of the Ras/MAPK cassette and the activated forms of the Drosophila EGF receptor (Egfr) and Sevenless (Sev) receptor tyrosine kinases (RTKs), which signal in the eye and wing, respectively. No dominant interaction is observed with sensitized conditions in the Wnt, Notch, Insulin-Pi3K, and DPP signaling pathways. The current hypothesis is that selenoproteins selectively modulate the Ras/MAPK signaling pathway through their antioxidant function. This is further supported by the fact that a selenoprotein-independent increase in ROS caused by the catalase amorphic Catn1 allele also reduces Ras/MAPK signaling. This study presents the first evidence for the role of intracellular redox environment in signaling pathways in the Drosophila organism as a whole (Morey, 2001).
The most striking result is that selD function modulates
the activity of Sev and Egfr RTKs, in the eye and wing
respectively, suppressing in transheterozygous combination
the phenotype caused by the gain-of-function mutations of these RTKs. In contrast, InR, another well-known RTK belonging to the insulin pathway, shows no modulation
by selDptuf mutation neither at the InR RTK level nor at
the level of the Pi3K. Therefore, selD somehow modulates
RTKs involved in the activation of the Ras/MAPK cassette
but not other RTKs. The suppression of the Raf gain-of-function
phenotype has been detected both in the eye and wing. A mild but statistically significant suppression of the rlSem
phenotype by selDptuf has been found in the eye but not in the wing. This is probably due to the different sensitivity
of both systems. The mild suppression of rlSem
phenotype could be due to the fact that the MAPK is the last step of the
pathway prior to the transcription of target genes. At this
point of the pathway, it may be difficult to block or dilute the amplified signal triggered by the rlSem gain-of-function mutation (Morey, 2001).
Surprisingly, no modulation of ras by selDptuf
mutation has been detected. There are two main explanations for this
result: (1) ras is not modulated by selD at all. The Sev, Egfr,
Raf and MAPK molecules, which are modulated by an
alteration of the redox balance, either by selDptuf
or Catn1 mutations, are kinases, whereas Ras is a GTPase. It is
possible that GPTases are not sensitive to changes in the
redox balance. This is supported by the fact that rasV12
rough eye phenotype is suppressed neither by selDptuf
nor Catn1 mutations. Similarly, the Rho1 Small-GTPase
rough eye phenotype is not suppressed by selDptuf
either. (2) ras is actually modulated by selD but beyond the
detection level, and therefore, no suppression can be scored.
It could also be that this modulation is towards an enhancement. It has been reported that 3T3 fibroblasts, when stably transformed with rasV12, produce large amounts of ROS superoxide (O22) by the activation of the NADPH oxidase
enzyme, which is triggered by the Rac signaling pathway. There is evidence that in certain cells MAPK activation in response to growth factors is dependent
on the production of ROS. Reduction of 50% of selD dose might be
not enough to alter the initial ras rough eye phenotype, neither to detect a suppression nor an enhancement. Besides, it must be kept in mind that Ras is a crosstalk point for other signaling pathways (such as Pi3K) and that the Raf/MAPK pathway is also activated in a Ras independent way (Morey, 2001).
With the exception of ras, a gradient was observed in
the strength of the suppression of phenotype has been observed, from sev being the strongest to rlSem
the weakest. This could be attributed to the differential sensitivity
of the particular gain-of-function alleles or to the
position in the pathway of the triggering gain-of-function element (Morey, 2001).
Homozygous selDptuf mutants lack selenoproteins and accumulate free radicals. Heterozygous selDptuf
individuals show no sign of impaired Ras/MAPK signaling due to the loss of
one dose of selD. However, when using activated elements
of the Ras/MAPK pathway, the effects of the lack of one
dose of selD are evident. Therefore, it is tempting to
speculate that selDptuf in heterozygosis results in an accumulation of ROS due to lower activity of the selenoproteins
biosynthesis. This accumulation would be sufficient to
impair the Ras/MAPK signaling, suppressing the gain-of-function
phenotypes of elements of the pathway. However,
the findings are in contrast with the results obtained in
tissue culture experiments. It has been observed that ligand
stimulation with peptide growth factors acting through
RTKs results in an increase in intracellular ROS. Moreover, ligand-stimulated ROS generation appears to have a role mediating tyrosine phosphorylation. In addition
to that, it has also been shown that extracellular administration of non-lethal concentrations of H2O2 activates MAPK. Altogether, these results in tissue culture systems point to activation of the pathway by ROS (Morey, 2001).
In this study, elimination or reduction of selenoprotein
function could result in prolonged activity of the signaling
pathway (due to the ROS increase caused by selDptuf) and
consequently induce apoptosis. Therefore, one could conclude
that the suppression observed in these experiments
could be due to cell death of the extra R7s. Loss of any cell
of the sevenless equivalence group by apoptosis will result in
rough eye phenotype. However, a rescue of the normal ommatidium organization,
which can only be achieved if the number of cells of the
ommatidium is not altered, has been demonstrated. For this reason, it is thought that apoptosis does not explain the strong suppression observed
in these experiments. Rather, these results point to a downregulation
of the pathway. The discrepancies found between cell
culture and Drosophila as a whole organism (i.e., activation
versus downregulation of the pathway by ROS respectively)
may be due to the inherent differences between the two
systems used. A more important difference, however, is
that experiments performed in tissue culture study the
effect of transient low concentration increases of ROS
whereas in this system a gene-dosage dependent constant increase in ROS occurs. To reconcile these discrepancies, it is proposed that transient increases in ROS
could have a physiological function on activation of phosphorylation
and thus trigger the Ras/MAPK signaling
pathway, whereas a constitutive pathological change in redox potential could activate a defense mechanism blocking the pathway (Morey, 2001).
This is the first example of the role of intracellular redox
environment on the Ras/MAPK signaling pathway in a
whole organism. The high specificity of these results (i.e., no
interaction with other signaling pathways and results
confirmed in two different developing tissues (wing and
eye) gives strong support to the notion that signaling
through the Ras/MAPK pathway is modulated by ROS. The
current hypothesis is that selenoproteins, through an undisclosed subset of ROS, modulate the Ras/MAPK signaling
pathway. This is consistent with the finding that this
pathway is also modulated by catalase. A selenoprotein-independent increase of a subset of ROS (i.e., H2O2) is able to modulate this pathway as well. This observation favors a scenario in which modulation of the Ras/MAPK pathway by selenoproteins would be achieved by their control of the redox balance rather than one in which selenoproteins would exert their role directly, interacting with one or more
elements of this signaling cassette. These results may help
to shed light on the role of redox on signaling events under
physiological conditions in multicellular organisms (Morey, 2001).
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 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).
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).
Retinoblastoma (Rb) family proteins control E2F-dependent transcription and restrict cell proliferation. In the early G1 phase of the cell cycle, Rb family proteins bind to E2F family members, inhibiting their ability to activate transcription and recruiting repressor complexes to DNA. In late G1 to S phase, cyclin-dependent kinases (CDK) phosphorylate Rb family proteins, liberating E2F and activating E2F-dependent transcription. One of the least-well-understood aspects of in vivo studies of Rb function is the fact that the inactivation of Rb often sensitizes cells to apoptosis.
The extent of apoptosis caused by the inactivation of Rb is highly cell type and tissue specific, but the underlying reasons for this variation are poorly understood. This study characterizes specific time and place during Drosophila development where rbf1 mutant cells are exquisitely sensitive to apoptosis. During the third larval instar, many rbf1 mutant cells undergo E2F-dependent cell death in the morphogenetic furrow. Surprisingly, this pattern of apoptosis is not caused by inappropriate cell cycle progression but instead involves the action of Argos, a secreted protein that negatively regulates Drosophila epidermal growth factor receptor (EGFR [DER]) activity. Apoptosis of rbf1 mutant cells is suppressed by the activation of DER, ras, or raf or by the inactivation of argos, sprouty, or gap1, and inhibition of DER strongly enhances apoptosis in rbf1 mutant discs. RBF1 and a DER/ras/raf signaling pathway cooperate in vivo to suppress E2F-dependent apoptosis and the loss of RBF1 alters a normal program of cell death that is controlled by Argos and DER. These results demonstrate that a gradient of DER/ras/raf signaling that occurs naturally during development provides the contextual signals that determine when and where the inactivation of rbf1 results in dE2F1-dependent apoptosis (Moon, 2006).
This study takes advantage of the observation that the inactivation of rbf1 in the Drosophila eye results in a distinctive pattern of apoptosis that is tightly linked to eye development, and this model system was used to define a cellular context in which RBF1 is needed to protect cells against dE2F1-dependent cell death. The results show that the cellular response to the inactivation of rbf1 involves a combination of signals. Deregulated dE2F1 provides one function that is required for apoptosis. However, in most situations, deregulation of the endogenous dE2F1 is not sufficient to induce apoptosis. In addition, a second condition, the down-regulation of an EGFR/Ras/Raf signaling pathway, is also necessary. In the eye imaginal disc, the EGFR/Ras/Raf signaling pathway is down-regulated at the region immediately anterior to the 'intermediate group' (IG) of cells, from which the R8 founder cell will be selected. When rbf1 mutant cells pass through this gradient, they become highly sensitive to dE2F1-dependent apoptosis. Elevation of the level of DER/Ras/Raf signaling by a variety of means suppresses apoptosis in rbf1 mutant cells. Conversely, expression of a dominant-negative mutant of DER strongly synergized with mutation of rbf1 to induce apoptosis (Moon, 2006).
Before starting this work, several different ways were considered in which the inactivation of RBF1 might result in apoptosis. If differentiated/differentiating cells try to reenter the cell cycle following the inactivation of RBF1, then an abnormal or inappropriate S-phase entry might cause apoptosis. Alternatively, one could argue that rapidly proliferating cells contain the highest levels of E2F transcriptional activity, and hence these cells ought to be most sensitive to E2F-induced apoptosis when RBF is removed. Although both models were plausible, in fact, neither explanation fits the data. rbf1 mutant eye discs display little apoptosis in either the population of differentiated cells or in actively cycling cells. Instead, rbf1 mutant cells are sensitive to apoptosis in the MF, at a time when some cells exit the cell cycle and initiate a differentiation program. This apoptosis was not accompanied by inappropriate cell cycle progression. Indeed, when rbf1 mutant cells were rescued from apoptosis, they showed no indication of S-phase entry. Hence rbf1 mutant cells were not dying because they were inappropriately progressing through the cell cycle. Instead, apoptosis was dependent on a specific developmental context. This need for the correct context may be particularly significant when designing cell culture-based screens for treatments that are synthetic lethal with the inactivation of Rb (Moon, 2006).
Several studies have shown that E2F complexes regulate the expression of proapoptotic genes, but why would the effects of losing RBF1 be sensitive to EGFR signaling? While it seems likely that deregulated dE2F1 activates transcription of several proapoptotic targets, the results indicate that an important part of the explanation lies in the regulation of the proapoptotic gene hid. hid transcripts are up-regulated in rbf1 mutant eye discs and halving the gene dosage of hid dramatically reduced apoptosis. Previous studies have shown that HID-induced apoptosis is highly sensitive to EGFR/Ras/Raf signaling. Signaling through this pathway suppresses transcription of hid and is thought to induce an inhibitory phosphorylation on the HID protein. This provides a simple model, in which the loss of RBF1 results in the elevated expression of a proapoptotic protein, which is then held in check by EGFR/Ras/Raf-mediated signaling. Apoptosis would then occur when EGFR signals are reduced. Consistent with this model, it was found that the region of the eye disc that is most sensitive to loss of RBF1 is also highly sensitive to low levels of ectopic hid expression (Moon, 2006).
Why does this pattern of apoptosis occur? It is likely that several different factors are needed to establish the gradient of DER activity. Important regulators of DER activity in the eye include Gap1, Sprouty, and Argos. In this particular context, the ability of Argos to diffuse and act at a distance from the p-Erk-positive cells appears to be important. It is suggested that the pattern of Argos expression in the developing eye disc generates a zone in which cells that have failed to exit the cell cycle and inappropriately inactivate RBF1 become prone to undergo apoptosis. In essence, this could be viewed as a developmental failsafe mechanism against inappropriate proliferation. In support of this, it is noted that E2F1 levels are transiently elevated in G1 phase cells in the MF, even though E2F regulation is not needed for S phase entry in the second mitotic wave. Consistent with the idea that this region of the disc may be more sensitive to apoptosis, it was found that a transient pulse of cyclin E expression, which drives ectopic S phases throughout much of the disc, generates a similar stripe of apoptosis in the MF. It is curious that this sensitivity occurs at the time when the role of EGFR is apparently changing from being needed for cell proliferation in the anterior part of the disc to being required for differentiation in the posterior part of the disc. It will be interesting to discover whether similarly sensitive regions exist in other discs (Moon, 2006).
As seen with rbf1, the effects of mutating Rb in the mouse are most evident at points in development when cells attempt to exit the cell cycle and differentiate. Rb-null mouse retinas show increased cell death during the transition from proliferation to differentiation. Whether this is due to an analogous interaction between Rb/E2F and EGFR/Ras signaling has not been tested but is an interesting possibility. It is also tempting to speculate that some of the different cellular responses to the inactivation of Rb in the mouse retina may be caused by differences in EGFR/Ras-mediated differentiation signals (Moon, 2006).
There are several indications that the general phenomenon described in this study is likely to be conserved in mammalian cells. For example, recent studies have shown that apoptosis in cultured fibroblasts lacking Rb family proteins (TKO) can be suppressed by activation of Ras/Raf. Interestingly, a functional homologue of Hid, Smac/Diablo, was recently shown to be a direct target of E2F1 in mammalian cells, raising the possibility that mammalian cells may contain a regulatory loop that directly parallels the regulation of Hid. However, a connection between the proapoptotic function of Smac/Diablo and EGFR pathway has yet to be described (Moon, 2006).
The molecular events underlying the convergence of EGFR signaling and Rb/E2F may be different between flies and humans. It is noted that Akt activation suppresses E2F1-induced apoptosis in mammalian tissue culture cells, while neither the overexpression of dAKT1 nor the mutation of dPTEN is sufficient to prevent cell death in rbf1 mutant eye discs. This may reflect a difference between an in vivo analysis and tissue culture conditions, or it may reflect species-specific differences in the regulation of apoptosis. It is known, for example, that caspase activation is regulated differently between species. In vertebrates, cytochrome c release from mitochondria is a key step in the promotion of caspase activation, while in Drosophila, this step is largely dispensable. It is possible that EGFR activity converges on E2F-dependent cell death through a previously identified E2F target whose activity is regulated by Raf/Erk- and/or AKT-mediated signals, such as Bim. In order to define this circuitry, it is first necessary to identify the appropriate in vivo context in mice or humans in which Rb/E2F and EGFR activity cooperate to regulate cell survival. Once the appropriate context is found, then it may be possible to identify the molecular mechanism linking E2F-dependent cell death to survival signals (Moon, 2006).
Both EGFR family and Rb pathways are often altered in cancer. Given that developmentally controlled fluctuations in EGFR signaling have dramatic effects on the sensitivity of rbf1 mutant cells to apoptosis, it is speculated that therapeutic cancer drugs that target EGFR family proteins may induce cell death most efficiently in tumor cells that have the highest levels of E2F1 activity. One of the curious features of human retinoblastoma is that, unlike many other cancers, these tumors rarely contain mutations in p53, suggesting that either these cells do not need to mutate p53 or that they find a more effective way to suppress apoptosis. Identification of the critical components that protect premaligant Rb mutant cells from apoptosis may lead to new ways to target these cells for treatment (Moon, 2006).
EGF receptor
:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions
| Developmental Biology
| References
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