Abstract

Tribbles homolog 2 (TRIB2) is critical for both solid and non-solid malignancies. Recently, TRIB2 was identified as a liver cancer-specific Wnt/β-catenin signaling downstream target and is functionally important for liver cancer cell survival and transformation. TRIB2 functions as a protein that interacts with E3 ubiquitin ligases and thereby modulates protein stability of downstream effectors. However, the regulation underlying TRIB2 protein stability per se has not yet been reported. In this study, we found that TRIB2 was up-regulated and exhibited high stability in liver cancer cells compared with other cells. We performed a structure-function analysis of TRIB2 and identified a domain (amino acids 1-5) at the N terminus that interacted with the E3 ubiquitin ligase Smurf1 and was critical for protein stability. Deletion of this domain extended TRIB2 half-life time accompanied with a more significant malignant property compared with wild type TRIB2. Furthermore, Smurf1-mediated ubiquitination required phosphorylation of TRIB2 by p70 S6 kinase (p70S6K) via another domain (amino acids 69-85) that is also essential for correct TRIB2 subcellular localization. Mutation of Ser-83 diminished p70S6K-induced phosphorylation of TRIB2. Moreover, the high stability of TRIB2 may be due to the fact that both p70S6K and Smurf1 were down-regulated and negatively correlated with TRIB2 expression in both liver cancer tissues and established liver cancer cell lines. Taken together, impaired phosphorylation and ubiquitination by p70S6K and Smurf1 increase the protein stability of TRIB2 in liver cancer and thus may be helpful in the development of diagnosis and treatment strategies against this malignant disease.

Highlights

  • Tribbles homolog 2 (TRIB2) is functionally important for liver cancer cell survival and transformation

  • TRIB2 Was Critical for Tumorigenesis in Liver Cancer Cells— First, we found that TRIB2 was highly up-regulated in liver cancer tissues compared with corresponding healthy tissues (Fig. 1A)

  • Treating cells with chemical inhibitors or stimulators of several key signaling cascades, we found that only treatment with the mammalian target of rapamycin signaling inhibitor rapamycin led to up-regulation of TRIB2 (Fig. 5A)

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Summary

Background

TRIB2 is functionally important for liver cancer cell survival and transformation. Results: Structure-function and biochemistry-based analysis revealed domains critical for TRIB2 protein stability. We performed a structure-function analysis of TRIB2 and identified a domain (amino acids 1–5) at the N terminus that interacted with the E3 ubiquitin ligase Smurf and was critical for protein stability. Deletion of this domain extended TRIB2 half-life time accompanied with a more significant malignant property compared with wild type TRIB2. Impaired phosphorylation and ubiquitination by p70S6K and Smurf increase the protein stability of TRIB2 in liver cancer and may be helpful in the development of diagnosis and treatment strategies against this malignant disease. Our study adds TRIB2 to the substrate list of Smurf and establishes the functional relationship between TRIB2 and p70S6K

EXPERIMENTAL PROCEDURES
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