Abstract
The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.
Highlights
The first tribbles protein was identified in Drosophila as a protein required for oogenesis [1]
By contrast when we compared these levels to arterial smooth muscle cells (ASMC), quite distinct results were obtained with levels of 1.3% departing significantly from the Protein Atlas value of 14% for smooth muscle
This report demonstrates that TRIB1 is an unstable protein expressed from an unstable transcript whose steady state can be increased by the proteasome, a major eukaryotic system for targeted intracellular protein degradation [17,36]
Summary
The first tribbles protein was identified in Drosophila as a protein required for oogenesis [1]. Higher eukaryotes express three distinct tribbles, TRIB1, 2 and 3 which are characterized by a relatively well conserved kinase-like domain (40–70% identity) and variable N and C-terminal regions [2]. Whether the tribbles are completely inactive as kinases is still unresolved as recent evidence suggests that TRIB2 possesses low nucleotide binding properties and a weak kinase activity at least in vitro, structural data suggest that this ability may be lost in TRIB1 [3,4]. Tribbles have been implicated in multiple types of cancer. Studies demonstrated that the tribbles are associated with myeloid leukemia [5,6] and a gain of function TRIB1 promotes.
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