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Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
RING finger-dependent ubiquitination by PRAJA is dependent on TGF-beta and potentially defines the functional status
1Department of Surgical Sciences, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20007, USA.
Abstract:
In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-beta signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-beta-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P < 0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Delta-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Delta-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-beta signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t(1/2)) and rate constant for degradation (k(D)) of ELF is 1.91 h and 21.72 min(-1) when coupled with ectopic expression of PRAJA in cells stimulated by TGF-beta, compared to PRAJA-transfected unstimulated cells (t(1/2) = 4.33 h and k(D) = 9.6 min(-1)). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.
Insights
High PRAJA expression in gastrointestinal cancers degrades ELF, an adaptor protein, disrupting TGF-beta signaling and potentially causing tumorigenesis through ubiquitin-mediated pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Transforming growth factor-beta (TGF-beta) signaling is crucial in gastrointestinal cells, involving transmembrane receptors and Smad proteins.
- Smad4, a tumor suppressor, is frequently mutated in gastrointestinal cancers, but its inactivation mechanism is unclear.
- ELF (embryonic liver fodrin) is a Smad4 adaptor protein whose disruption affects TGF-beta signaling.
Purpose of the Study:
- To investigate the role of PRAJA, a RING-H2 protein, in the TGF-beta signaling pathway and its potential involvement in gastrointestinal tumorigenesis.
- To elucidate the mechanism by which PRAJA affects ELF and Smad protein stability and function.
- To determine if PRAJA-mediated degradation of ELF contributes to alterations in TGF-beta signaling observed in cancer.
Main Methods:
- Investigated the interaction between PRAJA and ELF in gastrointestinal cancer cell lines.
- Assessed the effect of PRAJA expression on ELF and Smad protein levels and ubiquitination.
- Utilized stable cell lines overexpressing PRAJA and a mutant form (Delta-PRAJA) lacking the RING finger domain.
- Measured the half-life and degradation rate of ELF in hepatocytes with and without PRAJA expression under TGF-beta stimulation.
Main Results:
- PRAJA expression increased fivefold in gastrointestinal cancer cell lines, correlating with decreased ELF and Smad4 levels.
- PRAJA induced E3-dependent ubiquitination and degradation of ELF and Smad3, but not Smad4.
- Overexpression of PRAJA led to reduced ELF levels, while Delta-PRAJA overexpression maintained high ELF levels.
- Ectopic expression of PRAJA in hepatocytes significantly decreased ELF half-life and increased its degradation rate, particularly upon TGF-beta stimulation.
Conclusions:
- PRAJA enhances the degradation of ELF, a Smad adaptor protein, through the ubiquitin-mediated pathway.
- High PRAJA expression in gastrointestinal cancers likely contributes to tumorigenesis by disrupting TGF-beta signaling via ELF degradation.
- This study reveals a novel mechanism linking PRAJA, ELF degradation, and cancer development in the gastrointestinal tract.
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