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RING finger-dependent ubiquitination by PRAJA is dependent on TGF-β and potentially defines the functional status of the tumor suppressor ELF

Oncogene volume 25, pages 693–705 (2006)Cite this article

Abstract

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-β) 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-β signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-β-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. Δ-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 Δ-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-β 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 (t1/2) and rate constant for degradation (kD) of ELF is 1.91 h and 21.72 min−1 when coupled with ectopic expression of PRAJA in cells stimulated by TGF-β, compared to PRAJA-transfected unstimulated cells (t1/2=4.33 h and kD=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.

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Abbreviations

RING:

Really Interesting New Gene

TGF-β:

transforming growth factor-beta

ELF:

embryonic liver fodrin

SARA:

Smad anchor for receptor activation

ORF:

open-reading frame

HECT:

homologous to E6-AP C-terminus

HEF1:

human enhancer of filamentation 1

APC:

anaphase-promoting complex

IκB:

inhibitor of nuclear factor κB

PML:

promyelocytic leukemia protein

RFP:

ret finger protein

IAP:

inhibitors of apoptosis

PH:

partial hepatectomy

PBS:

phosphate-buffered saline

BSA:

bovine serum albumin

ATCC:

American Type Culture Collection

HCC:

hepatocellular carcinoma

DMEM:

Dulbecco's modified Eagle's medium

FBS:

fetal bovine serum

IP:

immunoprecipitation

IB:

immunoblotting

FITC:

fluorescein isothiocyanate

SDS:

sodium dodecyl sulfate

ECL:

enhanced chemiluminescence

WB:

Western blotting

SDS–PAGE:

SDS–polyacrylamide gel electrophoresis

VA-1:

ELF-specific polyclonal antibody

HA:

hemagglutinin

R-Smad:

receptor-regulated Smad

PDB:

protein data bank

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Acknowledgements

We thank Drs M Zasloff and S Evans for their critical review of the manuscript, and R Redman and A Rashid for their invaluable assistance with histopathology specimens and extensive confirmatory review of all slides. Grant Support: NIHR01 DK56111 (LM), NIHR01 CA106614-01A2 (LM), NIHR01 DK58637 (BM), VA Merit Award (LM), and R Robert and Sally D Funderburg Research Scholar (LM).

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Authors and Affiliations

  1. Departments of Surgical Sciences, Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA

    T Saha, D Vardhini, Y Tang, V Katuri, W Jogunoori, E A Volpe, A Sidawy, B Mishra & L Mishra

  2. Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA, USA

    D Haines

  3. Department of Cell Biology, Georgetown University, Washington, DC, USA

    X Zhou & I Gallicano

  4. Department of Pathology, Georgetown University, Washington, DC, USA

    R Schlegel

  5. Department of Veterans Affairs Medical Center, Washington, DC, USA

    A Sidawy & L Mishra

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  1. T Saha
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Correspondence to B Mishra or L Mishra.

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Saha, T., Vardhini, D., Tang, Y. et al. RING finger-dependent ubiquitination by PRAJA is dependent on TGF-β and potentially defines the functional status of the tumor suppressor ELF. Oncogene 25, 693–705 (2006). https://doi.org/10.1038/sj.onc.1209123

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