A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination - PubMed (original) (raw)

A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination

Natalia G Starostina et al. Dev Cell. 2007 Jul.

Abstract

In Caenorhabditis elegans, the Gli-family transcription factor TRA-1 is the terminal effector of the sex-determination pathway. TRA-1 activity inhibits male development and allows female fates. Genetic studies have indicated that TRA-1 is negatively regulated by the fem-1, fem-2, and fem-3 genes. However, the mechanism of this regulation has not been understood. Here, we present data that TRA-1 is regulated by degradation mediated by a CUL-2-based ubiquitin ligase complex that contains FEM-1 as the substrate-recognition subunit, and FEM-2 and FEM-3 as cofactors. CUL-2 physically associates with both FEM-1 and TRA-1 in vivo, and cul-2 mutant males share feminization phenotypes with fem mutants. CUL-2 and the FEM proteins negatively regulate TRA-1 protein levels in C. elegans. When expressed in human cells, the FEM proteins interact with human CUL2 and induce the proteasome-dependent degradation of TRA-1. This work demonstrates that the terminal step in C. elegans sex determination is controlled by ubiquitin-mediated proteolysis.

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Figures

Fig. 1

Fig. 1. FEM-1 associates with CUL-2 in C. elegans and interacts with the CBC core adapter ELC-1 through a VHL-box motif

(A) FEM-1 associates with CUL-2 in C. elegans. Anti-FLAG affinity purifications from wild type (left lane) or a strain expressing the cul-2::FLAG transgene (right lane). Purified proteins were separated by SDS-PAGE, stained with SYPRO Ruby, and identified by MALDI-TOF MS (indicated on the right). Immunoglobulin heavy chain, Ig(HCh); or light chain, Ig(LCh). (B) On the top is a schematic of FEM-1 that indicates N-terminal Ankyrin repeats (blue) and a C-terminal VHL-box (red). Below is an alignment of C. elegans and human VHL-box sequences from CBC SRSs. Residues conserved in at least half of the sequences are highlighted: orange, aliphatic residues; blue, polar; green, negatively charged; purple, positively charged; red, proline. A consensus of the C. elegans VHL-box is given above the sequences, with the following symbols for amino acid groups: φ, aliphatic; PO, polar; +, positively charged; −, negatively charged. α-helices in the human VHL-box are marked (h); residues that contact Elongin C (

E

for major and

e

for minor contacts) and CUL2 (C) (Stebbins et al., 1999). (C) FEM-1 interacts with ELC-1 through the VHL-box motif. FLAG-tagged wild-type FEM-1 (WT) or mutant FEM-1 8S (containing serines for the first eight amino acids of the VHL-box, LEQLLELD) were co-expressed in 293T cells with HA-tagged ELC-1. Anti-FLAG immunoprecipitations were analyzed by immunoblotting for HA-ELC-1 and FLAG-FEM-1. (D) FEM-1 binds ELC-1 in vitro. In vitro translated 35S-labeled FEM-1 was incubated with GST or GST-ELC-1 purified on glutathione beads, and bound protein was analyzed by SDS-PAGE/autoradiography (left). Input lane is 10% of the amount in the binding reaction.

Fig. 2

Fig. 2. cul-2 mutant males exhibit a feminization phenotype

(A) DIC images of wild-type (top) and cul-2(ek1) mutant (bottom) XO males. Images are composites of overlapping pictures. (B) DIC images of wild-type and cul-2 mutant male tails. Tail rays are numbered. Scale bars, 10 μm.

Fig. 3

Fig. 3. CUL-2 negatively regulates TRA-1 levels in the soma and germ line

(A) Simplified genetic pathway for C. elegans somatic sex-determination (Zarkower, 2006). Arrows indicate positive interactions and bars indicate negative interactions. (B-C) Anti-TRA-1 immunofluorescence staining of dissected adult intestine (B) and gonads (C) from the genotypes and sexes indicated. Note that germ cells in cul-2 mutants undergo a G1 phase arrest and are therefore larger than wild-type germ cells (Feng et al., 1999). Scale bars, 10 μm. (D) Graph of anti-TRA-1 signal derived from confocal microscopy of intestine (top) and distal germ cells (bottom). anti-TRA-1 signals (arbitrary units ± s.e.m.) for all genotypes/sexes were normalized to 100 a.u. for wild-type intestine nuclei.

Fig. 4

Fig. 4. TRA-1 isoforms accumulate in cul-2, fem-1, fem-2, and fem-3 loss-of-function mutants, and a tra-1 gain-of-function mutant

(A-C) Anti-TRA-1 western blots of whole-animal lysates of adults of the indicated genotypes and karyotypes. Anti-α-tubulin western blots are shown as loading controls. The location of full-length TRA-1A (FL), cleaved TRA-1A, and TRA-1B are marked by arrows and labeled on the right. Asterisk denotes a non-specific band. For all panels, the mutant alleles used are: loss-of-function cul-2(ek4), fem-1(hc17ts), fem-2(b245ts), fem-3(e1996) for XO, fem-3(e2006ts) for XX, and tra-1(e1099); and dominant, gain-of-function tra-1(e1575)/+. In tra-1(e1099) loss-of-function (lf) XX mutants, all three isoforms of TRA-1 are absent, while two non-specific protein bands remain, including a faint band that overlaps with the lower band of the TRA-1A full-length doublet.

Fig. 5

Fig. 5. The CBCFEM-1 complex with the cofactors FEM-2 and FEM-3 promotes TRA-1A degradation

(A) FEM-1-mediated degradation of TRA-1A. T7-TRA-1A was expressed with or without FLAG-FEM-1 in human 293T cells in the presence or absence of the proteasome inhibitor LLnL (listed at the top). Cell lysate and an immunoprecipitation with anti-FLAG antibody were analyzed by western blotting with anti-T7, anti-FLAG, and anti-human CUL2 antibodies. (B) C. elegans FEM-1 co-immunoprecipitates human CUL2 in 293T cells. (C) TRA-1A physically associates with CUL-2 in C. elegans when the proteasome is inactivated. CUL-2-FLAG was affinity purified from whole-worm extract of wild type (WT) or a him-8 mutant strain expressing the cul-2::FLAG transgene grown in the presence or absence of pbs-4 RNAi bacteria to inactivate the proteasome, and further analyzed by western blot with anti-TRA-1 (top) or anti-FLAG (bottom) antibodies. (D) Pulse chase experiment demonstrates that FEM-1 preferentially induces the degradation of the full-length TRA-1A isoform. A non-cleavable full-length T7-TRA-1A-nc mutant and a ‘pre-cleaved’ T7-TRA-1-N672 mutant (containing the N-terminal 672 residues) were co-expressed with FLAG-FEM-1 in the presence of LLnL. At time 0, LLnL was removed and further protein synthesis was blocked by cycloheximide. The final lanes (asterisk) are for a 48 hr time point without LLnL or cycloheximide. (E) The gain-of-function TRA-1-N86D protein is fully degraded only upon expression of all three FEM proteins. Combinations of the FEM proteins were co-expressed to determine their effect on TRA-1-N86D degradation. The last lane represents expression of all FEM proteins plus TRA-1-N86D in the presence of LLnL. The transfected tagged FEM proteins and human CUL2 were detected as expression and loading controls.

Fig. 6

Fig. 6. The three FEM proteins bind to TRA-1A and to each other in the context of a CBC complex

(A) Each of the three FEM proteins interacts with both TRA-1A and TRA-1-N86D. FLAG-FEM-1, VSVG-FEM-2, and V5-FEM-3 were co-expressed in 293T cells with either wild-type T7-TRA-1A or the gain-of-function T7-TRA-1-N86D in the presence of LLnL. The individual FEM proteins were immunoprecipitated and then analyzed by immunoblotting for T7-TRA-1. The top panel is a western of immunoprecipitates from cells co-expressing FEM and TRA-1A proteins; while the second panel shows immunoprecipitates from cells expressing TRA-1A proteins alone. Western blots of cell lysates are shown in the bottom two panels. (B) in vitro translated TRA-1A and FEM-1 bind recombinant GST-fusion FEM-2 and FEM-3. 35S-TRA-1A, 35S-TRA-1A-N86D, and 35S-FEM-1 were incubated with GST, GST-FEM-2, and GST-FEM-3 purified on glutathione Sepharose beads, and the beads were analyzed by SDS-PAGE/autoradiography. The middle panel is a Coomassie R250-stained gel showing the relative amounts of GST and GST-fusion proteins used in the reactions. (C) in vitro translated 35S-FEM-1 binds recombinant GST-TRA-1A in vitro. (D) Reciprocal co-immunoprecipitations of the FEM proteins co-expressed in 293T cells. Epitope-tagged FEM proteins (noted at the top) were co-expressed in 293T cells in double combinations or all together. Each FEM protein was immunoprecipitated (noted at the bottom) and analyzed by western blotting for co-precipitated proteins (noted on the left side). The last panels on the right are westerns of the lysates. (E) Co-immunoprecipitation of human and C. elegans CBCFEM-1 components. Anti-HA immunoprecipitation from 293T cells (treated with LLnL) co-expressing FLAG-FEM-1, VSVG-FEM-2, V5-FEM-3, and T7-TRA-1A with or without HA-ELC-1, followed by western blot to detect the tagged proteins and human CUL2. Actin was probed as a negative control. For (D) and (E), asterisk denotes immunoglobulin heavy chain.

Fig. 7

Fig. 7. FEM-2 and FEM-3 enhance CBCFEM-1-mediated ubiquitination of TRA-1A

(A) The ubiquitination of TRA-1A in 293T cells is stimulated by the FEM proteins. A non-cleavable gain-of-function TRA-1A (T7-TRA-1-N86D-nc) was co-expressed with HA-ubiquitin, and with or without FEM proteins (noted at the top). T7-TRA-1-N86D-nc was immunoprecipitated in the presence of 0.1% of SDS, followed by anti-HA and anti-T7 immunoblotting. Whole cell lysates were blotted to assess protein expression. (B)in vitro ubiquitination of TRA-1A by the CBCFEM-1 complex. Epitope-tagged ELC-1, FEM-1, and TRA-1A were co-expressed in 293T cells with or without FEM-2 and FEM-3 (noted at the top) in the presence of LLnL. The CBCFEM-1 complex was immunoprecipitated with anti-HA antibody (1st IP). The immunocomplex (with associated T7-TRA-1A) was used in an in vitro ubiquitination reaction. T7-TRA-1A was subsequently immunoprecipitated in the presence of 0.1% SDS (2nd IP), followed by immunoblotting for FLAG-ubiquitin and T7-TRA-1A. Samples from the 1st immunoprecipitation were analyzed by western blot with antibodies to the respective epitope tags and human CUL2 to reveal the level of proteins in the immunocomplex used in the ubiquitination reaction. (C) Model of CBCFEM-1 regulation of TRA-1A. (left) In XO males, the CBCFEM-1 complex is active and targets TRA-1A for degradation. FEM-1 binds full-length TRA-1A with the assistance of FEM-2 and FEM-3, which bind to each other and to TRA-1A. TRA-1A is polyubiquitinated by the complex and is subsequently degraded by the proteasome. In the absence of TRA-1A, genes that promote male fates are expressed. (right) In XX hermaphrodites, the ability of CBCFEM-1 to degrade TRA-1A is restricted [possible mechanisms include inhibitory binding of FEM proteins by the upstream regulator TRA-2 (Mehra et al., 1999), or post-translational modification of TRA-1A]. In the absence of degradation, the full-length TRA-1A protein is proteolytically processed to form the cleaved isoform, which represses genes that promote male fates.

Comment in

References

    1. Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with Image. J Biophotonics International. 2004;11:36–42.
    1. Ahringer J, Rosenquist TA, Lawson DN, Kimble J. The Caenorhabditis elegans sex determining gene fem-3 is regulated post-transcriptionally. Embo J. 1992;11:2303–2310. - PMC - PubMed
    1. Chen P, Ellis RE. TRA-1A regulates transcription of fog-3, which controls germ cell fate in C. elegans. Development. 2000;127:3119–3129. - PubMed
    1. Chin-Sang ID, Spence AM. Caenorhabditis elegans sex-determining protein FEM-2 is a protein phosphatase that promotes male development and interacts directly with FEM-3. Genes Dev. 1996;10:2314–2325. - PubMed
    1. Conradt B, Horvitz HR. The TRA-1A sex determination protein of C. elegans regulates sexually dimorphic cell deaths by repressing the egl-1 cell death activator gene. Cell. 1999;98:317–327. - PubMed

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