Abstract
The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(−) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.
Highlights
The Epidermal Growth Factor Receptor (EGFR)/Ras GTPase/Mitogen Activated Protein Kinase (MAPK) signal transduction pathway is evolutionarily conserved and is essential for animal development [1,2]
We found that rab-7(ok511) robustly suppressed the Vul phenotypes and induced a Muv phenotype in combination with let-23(sy1) and mutations in lin-2, lin-7, and lin-10 (Figure 2I and J, and than rab-7(ok511); lin-2(e1309) animals (Table 3))
We found that rab-7(ok511) failed to suppress the Vul phenotype of let-23(sy97) animals (Table 3), indicating that LET23 EGFR is required for the enhanced signaling in rab-7(ok511) mutants
Summary
The EGFR/Ras GTPase/Mitogen Activated Protein Kinase (MAPK) signal transduction pathway is evolutionarily conserved and is essential for animal development [1,2]. The EGFR is activated by ligand binding, which stimulates receptor dimerization, transautophosphorylation of cytoplasmic Tyrosine residues, and recruitment of phospho-Tyrosine binding proteins such as Grb. The binding of the Grb adaptor protein and the associated SOS protein (a Ras Guanine nucleotide Exchange Factor) to the EGFR results in the activation of the membrane associated Ras GTPase, and subsequently activation of the MAPK cascade consisting of Raf, MAPK/ERK Kinase (MEK) and Extracellular Regulated Kinase (ERK) [6]. EGFR and Grb recruit Cbl, an E3-ubiquitin ligase, which ubiquitinates Lysine residues on the EGFR, targeting it for lysosomal degradation [7]. The ultimate fusion of MVBs with the lysosome results in degradation of the EGFR. Since the EGFR can continue to signal from endosomes, regulators of endocytic trafficking are positioned to regulate the duration and strength of signaling
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