Arkadia activates Smad3/Smad4-dependent transcription by triggering signal-induced SnoN degradation.
E3 ubiquitin ligases play important roles in regulating transforming growth factor beta (TGF-beta)/Smad signaling. Screening of an E3 ubiquitin ligase small interfering RNA library, using TGF-beta induction of a Smad3/Smad4-dependent luciferase reporter as a readout, revealed that Arkadia is an E3 ubiquitin ligase that is absolutely required for this TGF-beta response. 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 show that Arkadia specifically activates transcription via Smad3/Smad4 binding sites by inducing degradation of the transcriptional repressor SnoN. Arkadia is essential for TGF-beta-induced SnoN degradation, but it has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-beta/Activin signaling, but SnoN is efficiently degraded only when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3. Finally, we describe an esophageal cancer cell line (SEG-1) that we show has lost Arkadia expression and is deficient for SnoN degradation. Reintroduction of wild-type Arkadia restores TGF-beta-induced Smad3/Smad4-dependent transcription and SnoN degradation in these cells, raising the possibility that loss of Arkadia function may be relevant in cancer.
Levy
Laurence
L
Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Howell
Michael
M
Das
Debipriya
D
Harkin
Sean
S
Episkopou
Vasso
V
Hill
Caroline S
CS
D000230:Adenocarcinoma; D000818:Animals; D001471:Barrett Esophagus; D002460:Cell Line; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; C060615:SKIL protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; D025801:Ubiquitin; C486067:RNF111 protein, human; C427283:Rnf111 protein, mouse; D044767:Ubiquitin-Protein Ligases