The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway

Abstract

Activation of the Raf kinase by GTP-bound Ras is a poorly understood step in receptor tyrosine kinase signaling pathways. One such pathway, the epidermal growth factor receptor (EGFR) pathway, is critical for cell differentiation, survival, and cell cycle regulation in many systems, including the Drosophila eye. We have identified a mutation in a novel gene, aveugle, based on its requirement for normal photoreceptor differentiation. The phenotypes of aveugle mutant cells in the eye and wing imaginal discs resemble those caused by reduction of EGFR pathway function. We show that aveugle is required between ras and raf for EGFR signaling in the eye and for mitogen-activated protein kinase phosphorylation in cell culture. aveugle encodes a small protein with a sterile ␣ motif (SAM) domain that can physically interact with the scaffold protein connector enhancer of Ksr (Cnk). We propose that Aveugle acts together with Cnk to promote Raf activation, perhaps by recruiting an activating kinase. Many receptor tyrosine kinases (RTKs), including the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), signal through the Ras/ mitogen-activated protein kinase (MAPK) pathway (Nishida and Gotoh 1993). These receptors have important developmental functions and are also misregulated in a variety of cancers (Holbro and Hynes 2004), making it critical to understand their signaling mechanism. Genetic screens in Drosophila and Caenorhabditis elegans, coupled with biochemical analysis in cultured cells, have allowed the identification of numerous Ras/MAPK pathway components (Rubin et al. 1997;Moghal and Sternberg 2003).The Drosophila eye is a particularly useful system for genetic analysis of the EGFR pathway, which has welldefined functions in photoreceptor development (Freeman 1997;Halfar et al. 2001;Yang and Baker 2003). As the morphogen Hedgehog (Hh) drives progression of the morphogenetic furrow across the eye disc, it induces the expression of the transcription factor Atonal, which specifies the first photoreceptor to differentiate in each cluster, R8 (Jarman et al. 1995;Dominguez 1999). Once the R8 photoreceptor has been specified, it sequentially recruits additional photoreceptors, cone cells, and pigment cells from the surrounding pool of undifferentiated cells. The signal for this recruitment is the EGFR ligand Spitz (Spi), which is secreted by R8 and subsequently by other photoreceptors as they differentiate (Freeman 1996). Production of the downstream feedback inhibitor Argos (Aos), which binds to Spi and blocks its binding to the receptor (Klein et al. 2004), restricts the response to Spi to a small number of cells, allowing the stepwise recruitment of ommatidial cell types (Freeman 1997). A second RTK, Sevenless, also contributes to R7 differentiation (Freeman 1996). The precursors of the R2, R5, R3, and R4 photoreceptors remain arrested in the G1 phase of the cell cycle after leaving the morphogenetic furrow. This arrest also requires EGFR signaling, but occurs at a lower threshold tha...

KSR and CNK: two scaffolds regulating RAS-mediated RAF activation

“…Functional characterization of HYP/AVE (referred to as HYP thereafter) indicated that it acts downstream of RAS, but at a step upstream of RAF. Furthermore, analysis of loss-offunction mutations confirmed its role downstream of EGFR during eye and wing development (Roignant et al, 2006). HYP presumably functions downstream of other RTKs as it has recently been identified in a cell-based genome-wide RNAi screen for genes required for ERK activation induced by various RTKs, including the Insulin receptor and a homolog of the plateletderived growth factor/vascular endothelial growth factor receptor (Friedman and Perrimon, 2006).…”

“…HYP presumably functions downstream of other RTKs as it has recently been identified in a cell-based genome-wide RNAi screen for genes required for ERK activation induced by various RTKs, including the Insulin receptor and a homolog of the plateletderived growth factor/vascular endothelial growth factor receptor (Friedman and Perrimon, 2006). A single homolog has been identified in C. elegans, whereas mammals contain at least two related proteins (Douziech et al, 2006;Roignant et al, 2006). Therefore, its function is presumably conserved in other metazoans.…”

“…Indeed, it was found to associate with a novel protein of 106 amino acids that essentially consists of a SAM domain. This protein has been independently identified by two groups and named hyphen (HYP) or aveugle (Ave) (Douziech et al, 2006;Roignant et al, 2006). Functional characterization of HYP/AVE (referred to as HYP thereafter) indicated that it acts downstream of RAS, but at a step upstream of RAF.…”

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation

“…Adult eye phenotypes thus provide a rapid screen for new regulators of the EGFR pathway (Legent et al, 2012;Miura et al, 2008;Roignant et al, 2006;Roignant and Treisman, 2010). Here, we report that a mutation in Vps4 disrupts EGFR signal transduction as well as the transduction of other signaling pathways.…”

“…Double-stranded RNAs (dsRNAs) were generated using the MEGAscript T7 and T3 Kits (Ambion) as described previously (Roignant et al, 2006) and 15 µg dsRNA were used to treat 10 6 cells per well for 5 days. EGFR expression was induced for 3 h with 60 µM Cu 2 SO 4 .…”