Many receptor tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), signal through the Ras/ MAPK pathway (Nishida
& Gotoh, 1993; Lusk et al., 2017). These receptors have important developmental functions and are also misregulated in a variety of cancers.
Figure 19: Diagrammatic representation of the EGF signaling pathway. EGFR ligands activated (vn, spi, krn) the pathway. Drk binds through its SH2 domain to the phosphorylated tyrosines on EGFR and in turn binds SOS which triggers activation of Ras. Ras activates Raf which phosphorylates MAPKK which phosphorylates MAPK which can phosphorylate pnt or yan which in turn lead to transcriptional responses, including the transcription of Arg which will down regulate the EGF pathway (adapted from Lusk et al., 2017).
The Drosophila homolog of EGFR is a single-pass transmembrane receptor tyrosine kinase (RTK) that transduces signal from a series of different ligands, which can interact with the receptor through different molecular mechanisms (Klein et al., 2008) (Figure 19). These ligands include gurken (grk), spitz (spi), keren (krn) that show homology to TGF-α and vein (vn), homologous to neuregulin (Paul et al., 2013; Steinhauer et al., 2013; Austin et al., 2014). In addition to these four extracellular ligands, Argos (arg) serves as a ligand antagonist by inhibiting EGF signal transduction (Klein et al., 2008).
Upon ligand binding, EGFR forms a dimer and trans-phosphorylates. DRK (Downstream of Receptor Kinase; the Drosophila homolog of mammalian Grb2) binds to the phosphorylated EGFR and is recruited at the plasma membrane. In turn, DRK binds Son of Sevenless (SOS), the guanine exchange factor, which triggers activation of RAS by promoting GTP binding. RAS activates RAF which phosphorylates MAPK kinases which phosphorylate rolled, a MAPK. Among many targets, activated rolled phosphorylates the transcription factors such as pnt and aop which in turn lead to transcriptional responses. In essence, the two transcription factors are in opposition with Pointed as the activator of EGF pathway target genes and aop as a repressor with edl balancing their function as seen before.
The first study that suggested a role of the MAPK pathway in Drosophila cellular immune response was done by Zettervall et al. (2004). To study the molecular mechanisms of this response, these authors have overexpressed different genes in the hemocytes, using the GAL4-upstream activating sequence system and a hemocyte-specific Hemese-Gal4 driver. They showed that surexpression of receptor tyrosine kinases, such as Egfr, Pvr, and Alk caused a drastic increase in the number of circulating hemocytes. An increase was also observed with the downstream signaling components Ras85D and pointed, supporting the notion that the Ras–MAPK pathway regulates hemocyte numbers. Pvr and Alk, also increased the lamellocyte production but not Egfr. The surexpression of an aop mutant protein (AopACT), with all possible MAPK phosphorylation sites mutated to acts as a constitutive repressor of Pointed, gives a massive lamellocyte response and a strong stimulation of hemocyte production instead of the antiproliferative effect expected. To explain this the authors suggested that different MAPKs may regulate a delicate balance between proliferation and lamellocyte activation by targeting different phosphorylation sites on aop and pnt.
Later, Egfr gene function in hemocytes in wild-type flies was suggested by Sinenko et al. (2012).
They showed that just after wasp parasitism an increase of ROS levels occurred in the PSC cells of the lymph gland, leading to the secretion of Spi, one ligand of the EGFR signaling pathway.
Figure 20: Proposed gene regulatory network that controls lymph gland rupture upon wasp parasitism. The PSC is drawn in grey. Wasp parasitism increases ROS in PSC cells that activate Toll/NF-kB and Spitz secretion (sSpi). Toll/ NF-kB activation in PSC cells requires SPE in the same cells for Spätzle processing (c-Spz). sSpi non cell-autonomously activates the EGFR pathway in lymph gland progenitors. Both EGFR and Toll/NF-kB activation are required for lymph gland lamellocyte differentiation, lymph gland disruption and wasp egg encapsulation. From (Louradour et al., 2017)
0 12 24 36 48
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The secretion of Spi into the hemolymph activates EGFR/Erk signaling in circulating hemocytes and triggers their differentiation into lamellocytes (Sinenko et al., 2012).
However, a direct role of EGFR activation in lymph gland progenitors was not rule out. More recently this was desmonstrated that the co-activation of EGFR signaling and Toll/NF-kB by ROS levels in the PSC controls the lymph gland hematopoiesis under parasitism (Louradour et al., 2017).
Toll/NF-kB signaling in PSC cells, while not required for lamellocyte differentiation, controls the timing of wasp-induced lymph gland dispersal and the release of lymph gland lamellocytes into circulation. The EGFR pathway activation in lymph gland progenitors is also required for on time lymph gland dispersal. The phosphorylation of ERK, undetectable in the lymph gland under normal conditions, increased in lymph gland progenitors 6h post-parasitism, indicating an EGFR activation. Furthermore, decreasing Spi expression in the PSC or down-regulating the EGFR pathway by expressing a dominant-negative form of the EGFR receptor in lymph gland progenitor cells, delays the lymph gland dispersal post-parasitism.
From all these data, the hypothesis is that edl and its interactors aop and pnt are implicated in the EGFR pathway activation in D. melanogaster hemocyte progenitors is required for their multiplication, differenciation and the on-time lymph gland dispersal (Figure 20).
Interestingly one of the phenotypic difference described between the Resistant (YR) and Susceptible (YS) fly strains is a different timing for the hemocyte increase in the hemolymph after parasitism by the Isy avirulent parasitoids (Russo et al., 2001) (Figure 21): the number of hemocytes in the R strain peaks at 15 h post-infestation and while it is 12h later for the YS strain. It is possible that this earlier ‘‘proliferation response’’ in the YR strain plays an important role for the encapsulation success.
At least, it is of note that TEL (translocation–Ets–leukemia or ETV6), the human aop ortholog, is required specifically for hematopoiesis within the bone marrow and its frequently found rearranged by chromosomal translocation in different human leukemias and cancers (Wang et al., 1998; Seth & Watson, 2005; Telford et al., 2016) In zebrafish, it has been shown that the TEL/ETV6 ortholog as several distinct roles for in embryonic hematopoiesis: etv6 knockdown resulted in reduced levels of progenitor cells, erythrocytes and macrophages (Rasighaemi et al., 2015).