The phylogenetic functional conservation ofDrosophilaSeven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, inDrosophilaeye development

Abstract

Key WordsSINA E3 ligase; ubiquitin-mediated proteolysis; RAS signaling transduction, Drosophila eye development, photoreceptor cell fate determination, and peripheral neuronal system development Abbreviations DmSINA PD -Drosophila SINA PD , DPP -decapentaplegic, EGFR -epidermal growth factor receptor, EMS -ethyl methanesulfonate, ERK -extracellular signal-regulated kinases, FLPrecombinase that recognizes FRT site, GAL4 -yeast DNA-binding transcriptional activator, GFP -green fluorescent protein, GMR -Glass multiple reporter, hSIAH1 PD -human SIAH1 PD , hSIAH2 PD -human SIAH2 PD , ORF -open reading frame, PNS -peripheral nervous system, PD -proteolysis deficient, RING -really interesting new gene, SEM -scanning electron microscopy, SEV -sevenless, SIAH -human homologs of SINA, SINA -seven in absentia, SINA/SIAH inhibitor -SINA PD /SIAH PD , UAS -yeast upstream activator sequence, WT -wild type Co-1 st authors Abstract Seven-IN-Absentia (SINA) is the most downstream signaling gatekeeper identified thus far in the RAS/EGFR pathway that controls photoreceptor cell fate determination in Drosophila.Underscoring the central importance of SINA is its phylogenetic conservation in metazoans, with over 83% amino acid identities shared between Drosophila SINA and human SINA homologs (SIAHs). SIAH is a major tumor vulnerability in multidrug-resistant and incurable cancer. SIAH inhibition is an effective strategy to shut down the tumor-driving K-RAS/EGFR/HER2 pathway activation that promotes malignant tumor growth and metastatic dissemination. To further delineate the SINA function in the RAS/EGFR pathway, a genetic modifier screen was conducted, and 28 new sina mutant alleles were isolated via ethyl methanesulfonate (EMS) and X-ray mutagenesis. Among them, 26 of the new sina mutants are embryonic, larval, or pupal lethal, and stronger than the five published sina mutants (sina 1 , sina 2 , sina 3 , sina 4 , and sina 5 ) which are early adult lethal. By sequencing the SINA-coding region of sina ES10 , sina ES26 , sina ES79 , and sina ES473 homozygous mutant animals, we identified three invariable amino acid residues in SINA's RING-domain whose single point mutation ablates SINA function. To demonstrate the functional conservation of this medically important family of RING domain E3 ligases in Drosophila, we established a collection of transgenic lines, expressing either wild type (WT) or proteolysis-deficient (PD) SINA/SIAH inhibitors of Drosophila SINA WT/PD and human SIAH1 WT/PD /2 WT/PD under tissue-specific GAL4-drivers in Drosophila eye, wing, and salary gland.Our results showed that Drosophila SINA and human SIAH1/2 are functionally conserved. Our bioengineered SINA PD /SIAH PD inhibitors are effective in blocking the RAS-dependent neuronal cell fate determination in Drosophila.