The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM-1 and FEM-3 for degradation by the proteasome

Abstract

The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and downstream of her-1 and probably tra-2. When expressed in mammalian cells, SEL-10 protein coimmunoprecipitates with FEM-1, FEM-2, and FEM-3, which are required for masculinization, and FEM-1 and FEM-3 are targeted by SEL-10 for proteasomal degradation. We propose that SEL-10-mediated proteolysis of FEM-1 and FEM-3 is required for normal hermaphrodite development.

Fig. 1.

The FEM proteins interact with SEL-10 in mammalian cells. Extracts from mammalian U2OS cells expressing SEL-10Myc; Flag-tagged FEM-1, -2, or -3; Flag-tagged TRA-2C; or both SEL-10Myc and the indicated Flag-tagged protein were immunoprecipitated by anti-Flag M2 or anti-Myc antibodies. The precipitated proteins were analyzed for the presence of SEL-10Myc with anti-Myc antibodies and the Flag-tagged proteins with anti-Flag M2 antibodies.

Fig. 2.

FEM-1 and FEM-3 may be targeted by hSEL-10 for degradation by the proteasome. To analyze protein steady-state levels, we treated BOSC cells expressing Flag-tagged FEM-1, -2 or -3, respectively, with lactacystin to inhibit the proteasome or with hsel-10 shRNA to partially inactivate hsel-10. The untreated and lactacystin-treated cells were cotransfected with a plasmid expressing control shRNA (firefly luciferase). Whole-cell lysates were analyzed by using anti-Flag M2 antibodies. Representative data from three independent experiments are shown.

Fig. 3.

Genetic and molecular pathways of somatic sex determination in C. elegans. (A) A simplified genetic pathway for sex determination in the C. elegans soma is shown. sel-10 is a new gene in this pathway and acts as a negative regulator of the fem genes. (B) A model for the molecular interactions among SEL-10, the FEM proteins, TRA-1, and TRA-2. SEL-10 negatively regulates FEM-1 and FEM-3 by promoting the degradation of their phosphorylated forms. A negative arrow from TRA-2 to FEM-3 reflects the possibility that TRA-2 directly binds and inhibits FEM-3 (7). See text for details.