Abstract
Simple SummaryTribbles proteins play various roles in cancer initiation and progression. However, still little is known about their molecular actions. Here we developed a mass spectrometry-based approach to study the Tribbles interactomes, allowing us to discover new interactors and functions that might help to understand their behavior better. Our proteomics data highlight the ability of TRIB3 to interact with transcription regulatory proteins and point to a new role in gene repression. Systematic analyses like these will help to evaluate the potential of Tribbles proteins as biomarkers for disease diagnosis and prognosis.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.
Highlights
Kinases regulate a plethora of cellular processes and changes in their enzymatic activity are intimately linked to human diseases, the large research field studying the basic biology of kinases and their potential as therapeutic targets [1,2]
As reported before [9,39], all Tribbles family members were able to physically interact with the E3 ubiquitin degradation complex formed by the ubiquitin E3 ligase COP1 and the adaptors proteins DET1 and DDB1
TRIB1 and TRIB2 were found to interact with TRIB1 and the Tribbles-related pseudokinase STK40, suggesting that Tribbles proteins can form homo- and heterodimers in mammalian cells, as has been shown for the Drosophila Tribbles homologue Trbl [40] and for the mammalian versions in protein complementation assays (PCA; unpublished observations)
Summary
Kinases regulate a plethora of cellular processes and changes in their enzymatic activity are intimately linked to human diseases, the large research field studying the basic biology of kinases and their potential as therapeutic targets [1,2]. While lacking intrinsic enzymatic activity, Tribbles proteins exert their biological roles predominantly via binding to other proteins, including kinases, phosphatases, transcription factors and components of the ubiquitin-proteosome system [14,15,16] This diverse range of interactors explains, at least in part, the difficulties to associate a TRIB family member with a single specific cellular pathway or role. TRIB3 has been shown to support breast and colorectal cancer stemness through the interaction with AKT and beta-catenin respectively [29,30] These examples illustrate that the different Tribbles family members can all play a regulatory role in cancer initiation and progression, but their contribution may be tumor type specific. We have generated an inducible system to evaluate the similarities and differences between TRIB1 and -3 interactomes in breast cancer cells as a first proof-of-principle study showing that comprehensive profiling of interactomes can improve our understanding of Tribbles’ role in cancer onset and progression
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