1. ¹Departments of Surgical Sciences, Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA
2. ²Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA, USA
3. ³Department of Cell Biology, Georgetown University, Washington, DC, USA
4. ⁴Department of Pathology, Georgetown University, Washington, DC, USA
5. ⁵Department of Veterans Affairs Medical Center, Washington, DC, USA

Correspondence: Dr B Mishra and L Mishra, Laboratory of Developmental Molecular Biology, Georgetown University, Medical–Dental Building, Rooms NW210–213, 3900 Reservoir Road, NW, Washington, DC 20007, USA. E-mail: bm72@georgetown.edu and lm229@georgetown.edu

Received 28 June 2005; Revised 11 August 2005; Accepted 11 August 2005; Published online 10 October 2005.

Abstract

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-) 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- signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF--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. -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 -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- 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-, 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.