PRAJA, an E3 ligase, may promote hepatocellular carcinoma through TGF-β dependent ubiquitination of ELF and Smad3, and via anti-apoptotic activity

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PRAJA, a RING-H2 finger E3 ubiquitin ligase, is up-regulated in many cancers including hepatocellular carcinoma (HCC). The transforming growth factor-beta (TGF-β) signaling pathway components Smad3 and embryonic liver fodrin (ELF), a type II β-spectrin, are ubiquitinated by PRAJA in a TGF-β dependent manner. ELF, which acts as a tumor suppressor for HCC, associates with Smad3 and Smad4 to transduce TGF-β signals to the nucleus. Therefore, PRAJA, through its ability to ubiquitinate ELF and Smad3, may influence critical TGF-β functions such as tumor suppression, cell differentiation and embryonic development. Here we investigated: 1) the role of PRAJA in embryonic development by anti-PRAJA Morpholino oligonucleotide based gene knockdown studies in zebrafish; 2) the role of PRAJA in liver formation by shRNA based loss-of-function studies in liver explant cultures; and 3) the molecular interactions between ELF, Smad3 and PRAJA in the presence and absence of TGF-β pathway activation, to elucidate the mechanism of PRAJA mediated TGF-β signaling and modulation of HCC. Confocal microscopy, co-immunoprecipitation analyses, luciferase reporter assays, and fos expression were used to reveal TGF-β dependent interactions between PRAJA and ELF, and between PRAJA and Smad3. Deletion constructs of PRAJA, ELF and Smad3 were used to demonstrate that, in the presence of TGF-β, ELF and Smad3 bind to the amino-terminal fragment of PRAJA (amino acids 1-150). Furthermore, the carboxy-terminal RING-H2 domain of PRAJA is required for TGF-β dependent ubiquitination of ELF and Smad3. Interestingly, loss of PRAJA function in liver explant tissue results in extensive cell death as well as activation of cartilage specific markers. Thus, PRAJA may regulate liver development, and inhibition of PRAJA could permit a switch to cartilage formation. Loss of PRAJA in zebrafish development results in a dramatic induction of apoptosis indicating that PRAJA has strong anti-apoptotic properties. In summary, these studies suggest that PRAJA promotes HCC through TGF-β dependent ubiquitination of ELF and Smad3, as well as through its anti-apoptotic activity.