We study how Smad proteins and bound cofactors assemble and read DNA, to correlate cell signaling with gene expression.
Cell fate is controlled by a multitude of signals and loss of this control has devastating consequences for living organisms. One of the key players in this network of signals is the TGF-beta family of cytokines. These hormones trigger an immense amount of responses by sending activated Smad transcription factors (Sma and Mad related proteins) to the nucleus where participate in many processes including the control of stem cell pluripotency and differentiation. At a molecular level, the basis for the transmission of information is the organization of complex networks orchestrated by Smads; many of which involve protein-protein and protein-DNA interactions.
Understanding how these SMAD complex networks function and how they are regulated requires efforts that combine functional approaches with the atomic resolution that structures can provide. To achieve these aims we have an ongoing collaboration with the group of Dr. J. Massagué at the Memorial Sloan-Kettering Cancer Center, NY.
According to their function, Smad proteins are classified as receptor regulated Smads (R-SMADs), which include Smads 1, 5 and 8 in the BMP-driven version of the SMAD pathway, and Smads 2 and 3 in the TGF-beta/ Nodal/Activin pathways. R-SMADs form complexes with the common co-activator Smad (Co-Smad), Smad4. The SMAD family also contains the two inhibitory Smads (I-Smads), Smad6 and Smad7, which provide critical negative regulation to these powerful and ubiquitous pathways.
In the last years we have determined how R- SMAD phosphorylation can lead to two distinct processes: activation (through interactions with YAP and Pin1 proteins) or ubiquitination (through interactions with Smurf1/2 and Nedd4L) and subsequent degradation. We found that degradation is a price that SMAD molecules pay for participating in transcription, with GSK3 modifying the phosphorylation code in the SMAD linker region from one that favors SMAD activation to one that favors SMAD destruction. As a result, TGF-beta/BMP signal delivery becomes coupled to SMAD turnover.
Smad7 recruits the ubiquitin ligases Nedd4L, Smurf1 and Smurf2 to mediate TGF beta receptor polyubiquitination and degradation by endocytosis. As with R-SMADS, all these interactions involve the linker region of Smad7 that contains a PY motif and the WW domain regions of Nedd4L, Smurf1/2, and YAP.
Our data reveal a surprisingly independence from phosphorylation in the interactions of Smad7, while phosphorylation is critical for the interaction of R-Smads, with YAP, Smurf1, Smurf2 and Nedd4L proteins. In addition, while pairs of WW domains recognize R-SMADs, binding to the inhibitor Smad7 requires the presence of single WW domains. These observations illuminate the functional versatility of WW domain containing proteins and also of the WW domains as mediators of specific interactions with SMAD proteins
NMR has been the main technique used to obtain structural information on proteins in the group for many years. As we moved to the study of larger protein systems, we have also introduced X-ray crystallography in our research. In addition, we have expanded our own knowledge and expertise to incorporate a variety of complementary techniques including modern molecular biology, peptide synthesis, peptide-protein native and chemical ligations, mass spectrometry (EM) and isothermal titration calorimetry (ITC).