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 (sina1, sina2, sina3, sina4, and sina5) which are early adult lethal. By sequencing the SINA-coding region of sinaES10, sinaES26, sinaES79, and sinaES473 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 SINAWT/PD and human SIAH1WT/PD/2WT/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 SINAPD/SIAHPD inhibitors are effective in blocking the RAS-dependent neuronal cell fate determination in Drosophila.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Financial Support: A.H.T and this work are supported by National Institutes of Health (R01 GM 069922, R01 CA140550, DOD-BCRP-BC095305, DOD-BCRP-BC0180907, and AACR-Pancreatic cancer action network innovative grant (#169458) to A.H.T.
Abbreviations
- DmSINAPD
- Drosophila SINAPD,
- DPP
- decapentaplegic,
- EGFR
- epidermal growth factor receptor,
- EMS
- ethyl methanesulfonate,
- ERK
- extracellular signal-regulated kinases,
- FLP
- recombinase that recognizes FRT site,
- GAL4
- yeast DNA-binding transcriptional activator,
- GFP
- green fluorescent protein,
- GMR
- Glass multiple reporter,
- hSIAH1PD
- human SIAH1PD,
- hSIAH2PD
- human SIAH2PD,
- 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
- SINAPD/SIAHPD,
- UAS
- yeast upstream activator sequence,
- WT
- wild type