y g by addition of nonrecombinant pCDNA3, as necessary. Results represent three t o six different transfected cultures, with the experiments performed on at least two different days. For dark conditions, plates of NIH 313 cells were wrapped in foil immediately after transfection until the time of harvesting (48 hours), except for a single brief medium change performed under dim red light (Kodak Safelight, Wratten Series 1A) 24 hours before harvesting. 18. E. A. Griffin Jr., D. Staknis, C. J. Weitz, unpublished data. (17) and light conditions were processed in parallel and incubated together. For light conditions, cells were illuminated (Fiber-Lite High Intensity Illuminator, Series 180; set at 10 relative illumination units) immediately after transfection until the time of harvesting. Plates were positioned 15 t o 30 cm from the tips of the twin fiberoptic guides. Transfected cells under dark

No abstract

The receptor-regulated Smad proteins are essential intracellular mediators of signal transduction by the transforming growth factor-beta (TGF-beta) superfamily of growth factors and are also important as regulators of gene transcription. Here we describe a new role for TGF-beta-regulated Smad2 and Smad3 as components of a ubiquitin ligase complex. We show that in the presence of TGF-beta signalling, Smad2 interacts through its proline-rich PPXY motif with the tryptophan-rich WW domains of Smurf2, a recently identified E3 ubiquitin ligases. TGF-beta also induces the association of Smurf2 with the transcriptional co-repressor SnoN and we show that Smad2 can function to mediate this interaction. This allows Smurf2 HECT domain to target SnoN for ubiquitin-mediated degradation by the proteasome. Thus, stimulation by TGF-beta can induce the assembly of a Smad2-Smurf2 ubiquitin ligase complex that functions to target substrates for degradation.