A screen for endocytic regulators of epidermal growth factor receptor signaling in Caenorhabditis elegans.

摘要

Epidermal Growth Factor Receptor (EGFR)/Ras/Mitogen Activated Protein Kinase (MAPK) signaling is involved in regulation of cell proliferation, migration and apoptosis. Activating mutations in EGFR or downstream components of the signaling pathway as well as a loss of a negative regulator of EGFR/Ras/MAPK signaling have been implicated in cancer. EGFR endocytosis and trafficking to the lysosome is an important mechanism of signal downregulation. A small GTPase Rab7 regulates EGFR trafficking and degradation, however its effects on signaling are not known.;To identify additional genes that might regulate LET-23 EGFR trafficking and signaling, I conducted a pilot forward genetic screen for suppressors of the lin-2(e1309) Vul phenotype. I identified two suppressor mutants, vh4 and vh22, that are partial embryonic lethal and display phenotypes suggestive of vesicular trafficking defects. I mapped vh4 and vh22 to distinct regions of chromosome I, devoid of previously identified regulators of LET-23 EGFR signaling, suggesting that they are mutations in novel regulators. Additionally, vh4 can enhance the Multivulva (Muv) phenotype of a Ras gain-of-function mutant let-60(n1046) further implicating vh4 as a negative regulator of EGFR/Ras/MAPK signaling. RNAi and genetic complementation data identified AGEF-1, an Arf-GEF, as a potential candidate for vh4. Although I have been unable to identify a corresponding mutation in agef-1, I find that vh4 mutants have phenotypes consistent of a defect in a secretory pathway and affecting AGEF-1 activity.;Thus, vh4 and vh22 might represent mutations in novel negative regulators of LET-23 EGFR signaling and possibly regulate trafficking of the receptor.;In C. elegans a highly conserved EGFR/Ras/MAPK signaling pathway is required for vulval cell fate specification. We found that RAB-7 antagonizes LET-23 EGFR signaling during vulval induction suggesting a possible role as a tumor suppressor in mammals. Here I show that a rab-7(ok511 ) deletion mutant regulates LET-23 localization in the Vulval Precursor Cells (VPCs). Additionally, my data suggest that LET-23 EGFR, similar to mammalian EGFR, traffics through multivesicular bodies (MVBs) en route to lysosome as RNAi knock-down of ESCRT-0 and --I components, HGRS-1 and VPS-28, can suppress the severity of the Vulvaless (Vul) phenotype of a LET-23 mislocalization mutant lin-2(e1309).