Analysis of human Tribbles 2 (TRIB2) pseudokinase
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Updated: Sep 24, 2025
Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
John A Harris1, Emma Fairweather1, Dominic P Byrne1
1Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Abstract:
Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.
Insights
Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase involved in cell survival and drug resistance. This study details lab procedures for purifying, stabilizing, and analyzing TRIB2, aiding the search for novel cancer therapeutics.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Human Tribbles 2 (TRIB2) is a pseudokinase implicated in cancer cell survival and drug resistance.
- Its precise physiological roles and therapeutic potential are not fully understood.
- TRIB2 interacts with key signaling modules like AKT, C/EBPα, and MAPK.
Purpose of the Study:
- To describe laboratory methods for the purification, stabilization, and analysis of human TRIB2.
- To outline screening procedures for identifying small molecule ligands targeting TRIB2.
- To facilitate further research into TRIB2 as a therapeutic target in cancer.
Main Methods:
- Purification of recombinant human TRIB2 protein.
- Stabilization techniques for pseudokinase analysis.
- Thermal shift assays for screening small molecule inhibitors.
- Analysis of TRIB2 interactions with E3 ubiquitin ligases.
Main Results:
- Established protocols for obtaining stable, purified human TRIB2.
- Demonstrated TRIB2's low affinity for ATP due to the absence of the DFG motif.
- Identified TRIB2 as a target for known small-molecule inhibitors.
- Characterized the regulatory role of the αC-helix and flanking region in TRIB2 stability.
Conclusions:
- Standardized laboratory procedures are crucial for studying TRIB2.
- TRIB2's unique structure presents opportunities for targeted drug development.
- Further investigation of TRIB2 ligands could lead to new cancer therapies.
