Analysis Of Tyrosine Phosphorylation Of Tumor Suppressor Smad4

The transforming growth factor-β (TGF-β) signalling pathway regulates cell functions and plays vital roles in development and tumor progression. Smad proteins are the intracellular effectors of TGF-β signaling that are activated by receptors, which consist of type II and type I receptors. Upon activation, Smads translocate into the nucleus, where they regulate transcription. Smad4, also known as co-Smad, is a key factor in both the TGF-β pathway and BMP pathway. As an intracellular signaling molecule, Smad4is regulated by posttranslational modifications such as serine/threonine phosphorylation, ubiquitination and sumoylation. To date, there have been no reports demonstrating tyrosine phosphorylation of Smad4.Protein tyrosine kinases (PTKs) are enzymes that phosphorylate specific tyrosine residues on protein substrates. Protein-tyrosine phosphorylation is an important mechanism for signal transduction. The human genome encodes90PTKs. These PTKs are involved in the regulation of growth, division, differentiation, survival, apoptosis and migration of cells.In this study, we explored possible tyrosine phosphorylation of Smad4. We sought to screen tyrosine kinases that can phosphorylate Smad4. We first established a mammalian expression library that contains82FLAG-tagged PTKs. Using combined biochemical approach of Co-Immunoprecipitation (Co-IP) and phospho-tyrosine-specific Western blotting, we found that10of the PTKs tested could phosphorylate Smad4. Among them, c-Abl kinase was chosen for further investigation because of its strong phosphorylation toward Smad4and their mutual interaction. c-Abl could localize both in cell cytoplasm and nucleus, and may play an important role in development of uncontrolled leukemia cell proliferation. In our study, we for the first time reported that c-Abl could specifically bind to and phosphorylate Smad4in a manner dependent on the kinase activity of c-Abl. By site mutagenesis, we identified the main tyrosine sites of Smad4phosphorylated by c-Abl. Further domain mapping identified the kinase domain of c-Abl as the main region for Smad4interaction and phosphorylation. Because Smad4is a transcription factor for TGF-β signaling, we conducted Luciferase Report Assay to further investigate the possible effects of c-Abl on TGF-β and BMP signaling pathways. Interestingly, results showed that c-Abl could inhibit responses in both pathways.In summary, this study has completed a cDNA-based screening for Smad4tyrosine kinases and chosen c-Abl as a representative kinase for in-depth analysis. For the first time we reported that c-Abl could interact with and phosphorylate Smad4. This study not only helps gain insights into understanding of tumor suppressor Smad4in tumorigenesis, but also has clinical implications in anti-cancer drug development.