Direct and heterologous approaches to identify the LET-756/FGF interactome

Abstract

Background: Fibroblast growth factors (FGFs) are multifunctional proteins that play important roles in cell communication, proliferation and differentiation. However, many aspects of their activities are not well defined. LET-756, one of the two C. elegans FGFs, is expressed throughout development and is essential for worm development. It is both expressed in the nucleus and secreted.

Results: To identify nuclear factors associated with LET-756, we used three approaches. First, we screened a two-hybrid cDNA library derived from mixed stages worms and from a normalized library, using LET-756 as bait. This direct approach allowed the identification of several binding partners that play various roles in the nucleus/nucleolus, such as PAL-1, a transcription regulator, or RPS-16, a component of the small ribosomal subunit. The interactions were validated by co-immunoprecipitation and determination of their site of occurrence in mammalian cells. Second, because patterns of protein interactions may be conserved throughout species, we searched for orthologs of known mammalian interactors and measured binary interaction with these predicted candidates. We found KIN-3 and KIN-10, the orthologs of CK2alpha and CK2beta, as new partners of LET-756. Third, following the assumption that recognition motifs mediating protein interaction may be conserved between species, we screened a two-hybrid cDNA human library using LET-756 as bait. Among the few FGF partners detected was 14-3-3beta. In support of this interaction we showed that the two 14-3-3beta orthologous proteins, FTT-1 and FTT-2/PAR-5, interacted with LET-756.

Conclusion: We have conducted the first extensive search for LET-756 interactors using a multi-directional approach and established the first interaction map of LET-756/FGF with other FGF binding proteins from other species. The interactors identified play various roles in developmental process or basic biochemical events such as ribosome biogenesis.

Figure 1

Co-immunoprecipitation of LET-756::GFP and HA-tagged protein partners Cos-1 cells were transfected with let-756::gfp alone (0) and with various HA-tagged constructs presumed or not (TACC1) to interact with LET-756. Twenty four hours later cell lysates were immunoprecipitated with anti-GFP antibodies. Western blots were first revealed with anti-HA antibodies and then with anti-GFP antibodies. An additional band, indicated with an asterisk, was consistently observed with HA- tagged RPL-6. A week and non- reproducible band can also be seen in the FTT-2 co-immunoprecipitation lane. None of these bands arose from possible complexes since 1) electrophoresis was performed in denaturating conditions and 2) antibody detection relied on the tag epitope and not on the endogenous protein.

Figure 2

Intracellular localization of LET-756::GFP and HA-tagged protein partners Cos-1 cells were transfected with let-756::gfp with or without various HA-tagged encoded constructs. Twenty four hours later cells were either fixed in 4% paraformaldehyde and permeabilized in triton or in cold methanol. Cells were incubated for an hour with rat anti-HA antibodies. Secondary goat anti-rat Texas red-coupled antibodies were then added for another 30 min. Coverslips were visualized with a confocal Leica microscope. Cells were transfected with the HA-tagged protein alone (column I) or cotransfected with let-756::gfp and revealed by direct fluorescence (column II), by anti-HA immunofluorescence (column III) and merge (column IV). In cells cotransfected with LET-756 and KIN-10 and treated with actinomycin D, both GFP staining and anti-HA immunostaining are displaced around and into the nucleolus. The yellow star indicates a neighbor single KIN-10 transfected cells. Its localization is not modified by the drugs. Panels A and B each show results for five interactors analyzed.

Figure 3

Interactome of the FGF family The identified interactors of various FGFs were grouped in six categories, depending of the functional activities of proteins. The human and C. elegans orthologs are indicated: human (white), worm (grey).