MAN1, an integral protein of the inner nuclear membrane, binds Smad2 and Smad3 and antagonizes transforming growth factor-beta signaling.
MAN1 (also known as LEMD3) is an integral protein of the inner nuclear membrane. Recently, mutations in MAN1 have been shown to result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. We show that the nucleoplasmic, C-terminal domain of human MAN1 binds to Smad2 and Smad3 and antagonizes signaling by transforming growth factor-beta (TGF-beta). In a yeast two-hybrid screen using the C-terminal domain of MAN1 as bait, eight positive clones were obtained that encoded Smad3. In direct two-hybrid assays, this portion of MAN1 bound to Smad2 and Smad3. In glutathione-S-transferase precipitation assays, the C-terminal domain of MAN1 bound to Smad2 and Smad3 under stringent conditions. Antibodies against MAN1 were able to co-immunoprecipiate Smad2 from cells, demonstrating that they reside in the same complex in vivo. TGF-beta treatment stimulated transcription from a reporter gene in control cells, but reporter gene stimulation was significantly inhibited in cells overexpressing MAN1 or its C-terminal domain but not its N-terminal domain. TGF-beta-induced cell proliferation arrest was also inhibited in stable cell lines overexpressing MAN1. These results show that the nuclear envelope regulates a signal transduction pathway and have implications for how mutations in nuclear envelope proteins cause different human diseases.
Lin
Feng
F
Department of Medicine and Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Morrison
Juliet M
JM
Wu
Wei
W
Worman
Howard J
HJ
D002460:Cell Line; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009685:Nuclear Envelope / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000502:physiology*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C405329:LEMD3 protein, human; D008565:Membrane Proteins; D009687:Nuclear Proteins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta