LB1
Arkadia function in the regulation of SnoN stability and its role as a tumour suppressor
Marco Briones-Orta, Laurence Levy, Caroline Hill
Cancer Research UK, London, UK
E3 ubiquitin ligases play important roles in regulating signaling by TGF-b superfamily members. We have screened an E3 ubiquitin ligase siRNA library, using TGF-b induction of a Smad3/Smad4-dependent luciferase reporter as a readout, and found that Arkadia is an E3 ubiquitin ligase that is absolutely required for this particular TGF-b response. We have shown that knockdown of Arkadia or overexpression of a dominant-negative mutant completely abolishes transcription from Smad3/Smad4-dependent reporters, but not from Smad1/Smad4-dependent reporters or from reporters driven by Smad2/Smad4/FoxH1 complexes. We have demonstrated that the mechanism whereby Arkadia specifically activates transcription via Smad3/Smad4 binding sites is through its ability to induce degradation of the transcriptional repressor, SnoN. We have shown that Arkadia is essential for TGF-b-induced SnoN degradation, but has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-b/Activin signaling, but SnoN is only efficiently degraded when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3.
Our data therefore indicate that loss of Arkadia results in stabilization of SnoN and loss of Smad3/Smad4-dependent transcription. Interestingly, both of these events can be features of human tumor cells. Consistent with this, we have found a cell line, SEG-1 that has lost Arkadia expression and is deficient for TGF-b-induced SnoN degradation. We have generated a SEG-1 stable cell line in which we reintroduced wild-type Arkadia. This restores Smad3/Smad4-dependent transcription and TGF-β-induced SnoN degradation. In contrast, neither the transcriptional or SnoN degradation responses are restored in a cell line that expresses an Arkadia-mutant in the RING-domain. Cell transformation assays using these cells have established that restoration of functional Arkadia inhibits the TGF-β-induced anchorage-independent growth seen in the parental line. Other preliminary results suggest that there are different rates of proliferation and migration between the lines expressing the wild-type and mutant Arkadia. The relevance of these results for tumorigenesis will be discussed.
