Comprehensive Profiling of Mammalian Tribbles Interactomes Implicates TRIB3 in Gene Repression

Abstract

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein-protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles' interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

Keywords: breast cancer; interactome; proteomics; tribbles.

Figure 1

TRIB1, -2 and -3 interactomes in HEK293T cells. (A) Schematic representation of workflow followed for AP-MS experiments. Figure was created with BioRender.com. (B) Volcano plots showing TRIB3 interactors compare to GFP control and TRIB3-mCOP1 interactors compared to WT TRIB3 in HEK293T cells. (C) Venn diagram of common and unique interactors between Tribbles family members. (D) TRIB3 interactors lost when comparing TRIB3 vs TRIB3-mCOP1 interactomes.

Figure 2

Contribution of TRIB3 domains to its interactome and function. (A) Human TRIB3 structure prediction by AlphaFold. Colors represent pLDDT score (blue: very high confidence, light blue: confident, yellow: low and orange: very low or unstructured). (B) Schematic representation of TRIB3 mutants and specific interactors lost when the indicated domain was removed. (C) Volcano plot showing interaction of the N-terminal and C-terminal domains of TRIB3 in HEK293T cells. (D) Co-immunoprecipitation assay of TRIB3-GFP and ZBTB1-Flag in HEK293T cells. (E) Gal4 reporter assay of Gal4-DBD, Gal4-TRIB3, Gal4-TRIB3-ΔN-terminal and Gal4-TRIB3-ΔC-terminal in Hek293T cells. Data is normalized using Renilla luciferase. (F) Western blot using Gal4DBD and tubulin antibodies showing similar expression of the constructs used for the Gal4 reporter assay.

Figure 3

TRIB1 and TRIB3 interactors in MCF7 cells. (A) Schematic representation of inducible constructs and workflow of the AP–MS experiments followed in MCF7 cells. (B) Western blot using t-GFP antibody sowing inducible expression of TRIB1-tGFP and TRIB3-tGFP upon doxycycline treatment and Tubulin expression as loading control. (C) Confocal images taken at 40× magnification showing tGFP, TRIB1-tGFP and TRIB3-tGFP localization upon doxycycline induction. (D) Volcano plots of TRIB1 and TRIB3 interactors compared with tGFP control in MCF7 cells and a volcano plot showing the comparison between TRIB1 and TRIB3 interactome in these cells. (E) Venn diagram of similar and different interactors between TRIB1 and TRIB3 in MCF7 cells detected in the AP–MSMS experiments. (F) Gal4 reporter assay of Gal4-DBD and Gal4-TRIB3 in MCF7 cells. Data is normalized using Renilla luciferase. Data is indicated as mean ± SEM. p-values were calculated using two-tailed Student’s t-test (* p < 0.05).