UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling inCaenorhabditis elegans

Abstract

Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells) to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components.

MIB-1 Is Required for Spermatogenesis and Facilitates LIN-12 and GLP-1 Activity in Caenorhabditis elegans

“…If wild-type MIB-1 acts to degrade APX-1 and/or ARG-1, one would expect glp-1(ar202gf) mib-1(lf) double mutants (which have no MIB-1 activity) to have an enhanced, not suppressed, Pro phenotype. Complicating any interpretation of Notch suppression is the fact that mib-1(lf) also suppresses glp-1(lf) in the embryo, but only under special conditions (20°growth) and not when the glp-1 and mib-1 lf phenotypes are most severe (25°growth conditions); this is similar to what has been previously found for the UBR-5 ubiquitin ligase (Safdar et al 2016). Collectively, these data indicate that mib-1(lf) likely suppresses lin-12(gf) and glp-1( gf) by some mechanism other than ligand degradation.…”

“…We created a glp-1(q231) mib-1(eb37) double mutant that showed no suppression of the Glp phenotype at a 25°growth temperature (Table 1), and these double mutants had a Glp germ line (data not shown). However, we did find that mib-1(eb37) significantly suppressed the embryonic lethal phenotype exhibited by glp-1(q231) mutants when they were grown at 20° (Table 1); at this growth temperature, this double mutant likely has partial gene function for both glp-1 (Safdar et al 2016) and mib-1. The glp-1(q231) mib-1(eb37) double mutant grown at 20°also shows a reduced number of dead eggs (Table 1), presumably because many, but not all, sperm produced by mib-1 mutants are defective at this growth temperature (data not shown).…”

“…For glp-1, mib-1(lf) can suppress the glp-1(ar202gf) proximal mitotic (Pro) phenotype and the embryonic lethal phenotype of glp-1(q231) when propagated at 20°. While lin-12(n302gf) mib-1(lf), glp-1(ar202gf) mib-1(lf), and glp-1(lf) mib-1(lf) double mutants all show incomplete penetrance (respectively, 20, 83, and 19% of individual worms show detectable suppression), this is typically the case for genetic suppression of either of these Notch paralogs (Tax et al 1997;Katic et al 2005;Katic and Greenwald 2006;Dunn et al 2010;Safdar et al 2016). In two of the cases, MIB-1 expression is detectable in the region (vulva precursor cells and proximal germ line) where the Notch paralog-associated phenotype is evident.…”

“…The second gene, sog-1, was identified as a suppressor of glp-1(q224ts) or glp-1(q231ts) loss-of-function mutations grown under semipermissive conditions Kimble 1989, 1993). This gene was subsequently shown to encode a homologous to the E6-AP carboxyl terminus (HECT)-type ubiquitin E3 ligase homologous to UBR-5 (Safdar et al 2016). ubr-5, like mib-1, has been evaluated in a glp-1(ar202) gain-of-function background.…”

“…While mib-1 loss-of-function mutations suppress the germ line phenotype caused by glp-1(ar202), ubr-5 loss-of-function mutations enhance glp-1(ar202)-associated tumor formation. So, the wild-type functions of the SEL-10 and UBR-5 ubiquitin E3 ligases are to downregulate Notch signaling, acting synergistically (Safdar et al 2016), while the wild-type MIB-1 ubiquitin E3 ligase somehow facilitates Notch-mediated signaling.…”